Recombinant hiv-1 envelope proteins and their use

ABSTRACT

HIV-1 Env ectodomain trimers stabilized in a prefusion mature closed conformation and methods of their use and production are disclosed. In several embodiments, the HIV-1 Env ectodomain trimers and/or nucleic acid molecules can be used to generate an immune response to HIV-1 in a subject. In additional embodiments, the therapeutically effective amount of the HIV-1 Env ectodomain trimers can be administered to a subject in a method of treating or preventing HIV-1 infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 16/557,894,filed Aug. 30, 2019, which is a continuation of U.S. patent applicationSer. No. 15/508,885, filed Mar. 3, 2017, now U.S. Pat. No. 10,400,015,which is the U.S. National Stage of International Application No.PCT/US2015/048729, filed Sep. 4, 2015, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application Nos. 62/046,059, filed Sep. 4, 2014, and62/136,480, filed Mar. 21, 2015. Each of the above-listed applicationsis incorporated herein by reference in its entirety.

FIELD

This disclosure relates to recombinant human immunodeficiency virus type1 (HIV-1) envelope (Env) polypeptides and immunogenic fragments thereoffor treatment and prevention of Human Immunodeficiency Virus (HIV)infection and disease.

BACKGROUND

Over the last 50 years, millions of people have been infected or killedby HIV-1. A dominant contributing factor has been the immunoevasion ofthe HIV-1-Env ectodomain trimer, a type 1 fusion machine thatfacilitates virus entry into cells by interacting with host cellularreceptors and fusing virus and host-cell membranes. Despite its exposedposition on the viral membrane and high titers of Env-reactiveantibodies in infected individuals, the HIV-1-Env ectodomain trimersuccessfully evades most antibody-mediated neutralization. This evasionis to a large degree responsible for the difficulty in developing aneffective HIV-1 vaccine.

The HIV-1-Env ectodomain trimer includes three gp120-gp41 protomers, anddisplays unusual posttranslation processing including the addition of25-30 N-linked glycans per gp120-gp41 protomer, tyrosine sulfation, andslow signal peptide cleavage. As an entry machine, HIV-1 Env undergoesseveral structural rearrangements from a prefusion mature (cleaved)closed conformation that evades antibody recognition, throughintermediate conformations triggered by CD4 and co-receptor (either CCR5or CXCR4) binding, to a postfusion conformation. Over the last 20 yearssubstantial atomic-level detail has been obtained on theseconformations, including structures of CD4-bound gp120, postfusion gp41,and the trimeric arrangement of prefusion gp120. The prefusion matureclosed conformation of HIV-1 Env has, however, resisted atomic-levelanalysis.

It is believed that immunization with an HIV-1 Env ectodomain trimerstabilized in its prefusion mature closed conformation can elicit aneutralizing immune response that is protective against HIV infection.However, the lack of an atomic-level structure of the HIV-1 Envectodomain trimer in this conformation stymied attempts to design HIV-1Env proteins that are stabilized in a prefusion mature closedconformation for use as immunogens.

SUMMARY

Disclosed herein for the first time is the atomic-level threedimensional structure of the HIV-1 Env ectodomain (including gp120 andthe extracellular portion of gp41) in its prefusion mature closedconformation, the conformation of Env recognized by most broadlyneutralizing antibodies. When viewed in the context of previouslydetermined structures of HIV-1 Env in a CD4-bound conformation, the newstructure provided herein affords a mechanistic understanding of theconformational transitions that HIV-1 Env undergoes from the matureclosed to CD4-bound open prefusion conformations. Analysis of theseconformational rearrangements, combined with an understanding of theevasion from and vulnerabilities to the immune system, provided aninformation matrix that was used to generate recombinant HIV-1 Envectodomain trimers stabilized in the prefusion mature closedconformation.

The disclosed recombinant HIV-1 Env ectodomain trimers resist transitionto the CD4-bound open conformation of HIV-1 Env when incubated with CD4,and thus will retain the prefusion mature closed conformation when usedas an immunogen to generate an HIV-1 Env immune response in a subjectexpressing CD4, such as a human. Retention of the prefusion matureclosed conformation in the presence of CD4 avoids exposure of highlyantigenic sites on the open conformation of the HIV-1 Env ectodomainthat are targeted by poorly neutralizing antibodies (such as 447-52D),and maximizes exposure of antigenic sites on the V1V2 “cap” of the HIV-1ectodomain that are targeted by broadly neutralizing antibodies.

In several embodiments, an isolated immunogen is provided that comprisesa recombinant HIV-1 Env ectodomain trimer or immunogenic fragmentthereof stabilized in a prefusion mature closed conformation by one ormore amino acid substitutions compared to a native HIV-1 Env sequence.The recombinant HIV-1 Env ectodomain comprises three gp120-gp41protomers comprising a gp120 polypeptide and a gp41 ectodomain, andremains in the prefusion mature closed conformation when incubated witha molar excess of soluble CD4 (sCD4). In several embodiments, therecombinant HIV-1 Env ectodomain trimer comprises an α7 helix that formsafter position 570 of the gp41 ectodomain, and comprises V1V2 domainswherein the distance between positions 200 and 313 of adjacent V1V2domains in the ectodomain trimer is less than five angstroms. Inadditional embodiments, the recombinant HIV-1 Env ectodomain trimer orimmunogenic fragment specifically binds to VRC26 mAb and/or PGT145 mAb,and does not specifically bind to 17b mAb when incubated with a molarexcess of sCD4.

In some embodiments, the recombinant HIV-1 Env ectodomain can bestabilized in the prefusion mature closed conformation by a non-naturaldisulfide bond between cysteine substitutions at positions 201 and 433.In additional embodiments, the recombinant HIV-1 Env ectodomain can bestabilized in the prefusion mature closed conformation by a non-naturaldisulfide bond between cysteine substitutions at positions 201 and 433,a non-natural disulfide bond between cysteine substitutions at positions501 and 605, and a proline substitution at position 559. The HIV-Envpositions correspond to a HXB2 reference sequence set forth as SEQ IDNO: 1. The recombinant HIV-1 Env ectodomain can comprises the sequencefrom a native HIV-1 Env ectodomain, such as from any one of a BG505 (SEQID NO: 2), CAP256.SU (SEQ ID NO: 51), a BB201.B42 (SEQ ID NO: 81), aKER2018.11 (SEQ ID NO: 107), a CH070.1 (SEQ ID NO: 174), a ZM233.6 (SEQID NO: 745), a Q23.17 (SEQ ID NO: 746), a A244 (SEQ ID NO: 747), aT250-4 (SEQ ID NO: 2114), 426c (SEQ ID NO: 2144), 45_01dG5 (SEQ ID NO:2145), JRFL (SEQ ID NO: 2115), or a WITO.33 (SEQ ID NO: 748) strain ofHIV-1, that has been modified to include the amino acid substitutionsthat stabilize the recombination HIV-1 Env ectodomain in the prefusionmature closed conformation.

In some embodiments, the recombinant HIV-1 Env ectodomain trimer can bea chimeric HIV-1 Env ectodomain trimer that comprises amino acidsequences from two or more HIV-1 strains. The use of immunogens based ondiverse HIV-1 strains can overcome the intrinsic sequence diversity ofHIV-1 Env. For example, the recombinant HIV-1 Env ectodomain trimer cancomprise a V1V2 domain (such as HIV-1 positions 126-196) from a firststrain of HIV-1, with the remainder of the recombinant HIV-1 Envectodomain trimer from a heterologous strain of HIV-1. In one example,the recombinant HIV-1 Env ectodomain trimer can comprise a V1V2 domainsequence from any one of a CAP256.SU (SEQ ID NO: 51), a BB201.B42 (SEQID NO: 81), a KER2018.11 (SEQ ID NO: 107), a CH070.1 (SEQ ID NO: 174), aZM233.6 (SEQ ID NO: 745), a Q23.17 (SEQ ID NO: 746), a A244 (SEQ ID NO:747), a T250-4 (SEQ ID NO: 2114), or a WITO.33 (SEQ ID NO: 748) strainof HIV-1, with the remainder of the recombinant HIV-1 Env ectodomainsequence from the BG505 (SEQ ID NO: 2) strain of HIV-1. The chimericHIV-1 Env ectodomain trimer further includes the one or more amino acidsubstitutions compared to a native HIV-1 Env sequence for stabilizationin the prefusion mature closed conformation. In some embodiments, thechimeric HIV-1 Env ectodomain trimer can comprise a non-naturaldisulfide bond between cysteine substitutions at positions 201 and 433,a non-natural disulfide bond between cysteine substitutions at positions501 and 605, and a proline substitution at position 559, to stabilizethe HIV-1 Env trimer in the prefusion mature closed conformation.

As described in the Examples, prefusion mature gp41 wraps itshydrophobic core around extended N- and C-termini-strands of gp120.Accordingly, in some embodiments, the recombinant HIV-1 Env ectodomaintrimer can include a membrane proximal “platform” including the N- andC-terminal regions of gp120, and the gp41 ectodomain, from a first HIV-1strain (such as BG505), and the remainder of gp120 from one or moreheterologous HIV-1 strains. This chimeric design allows for productionof heterogeneous HIV-1 Env proteins that comprise membrane distalfeatures of interest (such as the V1V2 domain, V3 domain, and CD4binding site) from diverse strains. In some embodiments, the recombinantEnv ectodomain can include gp120 residues 31-45 and 478-507, and thegp41 ectodomain (e.g., positions 512-664) from the first HIV-1 strain(such as BG505), and the remainder of the gp120 residues in the Envprotein can be from a heterologous HIV-1 strain. In some embodiments,the heterologous HIV-1 strain can be selected from one of a CAP256.SU(SEQ ID NO: 51), a BB201.B42 (SEQ ID NO: 81), a KER2018.11 (SEQ ID NO:107), a CH070.1 (SEQ ID NO: 174), a ZM233.6 (SEQ ID NO: 745), a Q23.17(SEQ ID NO: 746), a A244 (SEQ ID NO: 747), a T250-4 (SEQ ID NO: 2114),426c (SEQ ID NO: 2144), 45_01dG5 (SEQ ID NO: 2145), a JRFL (SEQ ID NO:2115) or a WITO.33 (SEQ ID NO: 748) strain of HIV-1. The chimeric HIV-1Env ectodomain trimer further includes the one or more amino acidsubstitutions compared to a native HIV-1 Env sequence for stabilizationin the prefusion mature closed conformation. In some embodiments, thechimeric HIV-1 Env ectodomain trimer can comprise a non-naturaldisulfide bond between cysteine substitutions at positions 201 and 433,a non-natural disulfide bond between cysteine substitutions at positions501 and 605, and a proline substitution at position 559, to stabilizethe HIV-1 Env trimer in the prefusion mature closed conformation.

In some embodiments, the gp120-gp41 protomers in the recombinant HIV-1Env ectodomain trimer can be single chain HIV-1 Env ectodomains, whereinthe C-terminal residue of the gp120 polypeptide is linked to theN-terminal residues of the gp41 ectodomain (for example, by a peptidelinker, such as a 10 amino acid or 15 amino acid glycine-serine peptidelinker). In some embodiments, the recombinant HIV-1 Env ectodomaintrimer can be linked to a transmembrane domain. For example, theC-terminal residue of gp41 (such as position 664) can be lined to atransmembrane domain by a peptide linker, such as a 10 amino acidglycine-serine peptide linker. The transmembrane domain can be, forexample, an influenza HA transmembrane domain.

In additional embodiments, the recombinant HIV-1 Env ectodomain trimerstabilized in the prefusion mature closed conformation can be includedon a protein nanoparticle, such as a ferritin or lumazine synthaseprotein nanoparticle. Nucleic acid molecules encoding the recombinantHIV-1 Env ectodomain trimer stabilized in the prefusion mature closedconformation and vectors including the nucleic acid molecules are alsoprovided. Compositions including the recombinant HIV-1 Env ectodomaintrimer or immunogenic fragments thereof, protein nanoparticles, nucleicacid molecules or vectors are also provided. The composition may be apharmaceutical composition suitable for administration to a subject, andmay also be contained in a unit dosage form. The compositions canfurther include an adjuvant. The recombinant HIV-1 Env ectodomaintrimers may also be conjugated to a carrier to facilitate presentationto the immune system.

Methods of generating an immune response in a subject are disclosed, asare methods of treating, inhibiting or preventing a HIV-1 infection in asubject. In such methods a subject, such as a human subject, isadministered an effective amount of a disclosed recombinant HIV-1 Envectodomain trimer or fragment thereof, protein nanoparticle, nucleicacid molecule or viral vector.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate the structure of a prefusion HIV-1-Env trimerbound by PGT122 and 35O22 antibodies. FIG. 1A, smFRET of functionalviral ectodomains, unliganded or in the presence of antibodies PGT122and 35O22. Fluorophores were introduced into the V1 and V4 regions ofJR-FL gp120, and smFRET signals measured with HIV-1 Env in themembrane-bound virion context (see FIG. 7 ). The concordance betweenconformational ensembles indicates unliganded and PGT122+35O22-boundconformation to be similar (Spearman correlation coefficient of 0.988).FIG. 1B, Ternary complex structure of HIV-1-Env trimer BG505 SOSIP.664with PGT122 Fab and 35O22 Fab. The gp120 subunit is shown in dark grey,the gp41 subunit in lighter grey. One protomer and associated Fabs isshown in ribbon and stick representation, a second protomer in surfacerepresentation, and the third protomer in grey.

FIGS. 2A-2C illustrate the prefusion structure of gp41. FIG. 2A, gp41forms a 4-helix collar which wraps around extended N and C termini ofgp120. Both gp120 (darker grey) and gp41 (lighter grey) are depicted inribbon representation, with select residues and secondary structurelabeled. The orientation shown here is similar to that of FIG. 1C, withperpendicular orientations provided in FIGS. 2B and 2C. (insert). Thegp41 collar is clasped by the insertion of Met530gp41 into a tryptophansandwich and by the complementary dipoles of helices α6 and α8.2F_(o)-F_(c) electron density for clasp residues is depicted at Is. FIG.2B, gp41 holds the N and C termini of gp120 in its hydrophobic core.Representation are the same as in a, excepted that hydrophobic sidechains are shown in stick representation and the orientation is rotated90° to depict the view from the viral membrane. FIG. 2C, gp41-trimerinterfaces as viewed from side in ribbon and surface representation.

FIGS. 3A-3D illustrate entry rearrangements of HIV-1 Env. FIG. 3A, BG505sequence of gp41 subunit (positions 512-664 of SEQ ID NO: 2), withprefusion and postfusion secondary structure. Fusion peptide (FP) isunderlined. FIG. 3B, Difference distance analysis of prefusion BG505 andpostfusion HIV-1/SIV chimeric gp41. Missing residues of BG505 (548-569)and of SIV (611-614) are indicated, along with secondary structure. FIG.3C, Superposition of postfusion gp41 onto prefusion gp41 for α7 (left)and α9 (right) prefusion helices. FIG. 3D, HIV-1-Env entryrearrangements. EM reconstructions (top row) with gp120 (middle) andgp41 (bottom) rearrangements between each conformational statehighlighted with lines to depict moving Cα between each conformation.Subunit models are shown in gray with modeling parameters provided inFIG. 15 . Antigenic recognition of each of these state is shown in FIG.26 .

FIGS. 4A-4C illustrate that the prefusion HIV-1 gp120-gp41 structureshares conserved structural and topological features with other type 1fusion machines. FIG. 4A, Prefusion (left) and postfusion (right)structures. The prefusion structures are shown for a single protomer inribbon-representation. FIG. 4 B, the preformed C-terminal helix ofpostfusion coiled coil from a is shown, with fusion peptides (FP) and Nand C terminal residues of postfusion coiled coils labeled, and thedistance the inner coiled coil extends between prefusion and postfusionconformations indicated. FIG. 4C, The gp41-equivalents encircle extended3-strands of their gp120-equivalent partners. Ribbon representation areshown looking towards the viral membrane.

FIGS. 5A-5C illustrate the fully assembled shield revealed by prefusionHIV-1 gp120-gp41 trimer. FIG. 5A, Glycan shield. Env N-linked glycansare depicted in light grey (conserved; greater than 90% conservation) ordark grey (variable; less than 90% conservation) on the maturenear-native Env structures for BG505 strain of HIV-1 (left), influenzavirus H3 hemagglutinin (PDB: 2YP7) (middle), and RSV fusion glycoproteinsubtype A (PDB: 4JHW) (right). Two conserved glycans, at residues241gp120 and 616gp41 are not in the BG505 sequence. FIG. 5B, Envsequence variability. FIG. 5C, HIV-1-Env N-linked glycans and sequencevariability for near-native and CD4-bound conformations.

FIGS. 6A-6E illustrate the location and prevalence on the HIV-1-Envectodomain trimer of neutralizing responses identified serologicallyfrom cohorts from 2-3 and 5+ years post-infection. FIG. 6A, The locationof the neutralization epitopes for the different antibody specificitieson the prefusion mature closed Env ectodomain trimer is depicted (top),with CD4-binding-site-directed antibody specificities (VRC01-, b12-,CD4-, and HJ16-like), 8ANC195-like, PG9-like (V1V2-directed), glycan V3specificities (PGT128- and 2G12-like), 35O22-like specificities, andPGT151-like specificities indicated. FIG. 6B, (top) Broadly neutralizingepitopes on influenza virus hemagglutinin (left) and RSV fusionglycoprotein (right). (bottom) Glycan surface area and residue entropyin antibody epitopes for HIV-1, influenza, and RSV. FIG. 6C,Neutralization fingerprint. For each serum, the predicted neutralizationprevalence for each of the 12 antibody specificities is shown based onneutralization of 21 diverse HIV-1 strains. FIG. 6D, Prevalence ofantibody specificities onto the HIV-1-Env. FIG. 6E, Antibodyspecificities with high serum prevalence in the 5+ years cohort aredepicted with antigen-binding fragments of representative antibodies(surface transparency proportional to prevalence) on the BG505 Envtrimer, which is shown in cartoon representation, with glycans assticks.

FIGS. 7A-7D show smFRET of HIV-1 Env in the context of infectious JR-FLvirions. HIV-1_(JR-FL) gp160 was labelled with fluorescent dyes invariable regions V1 and V4 at positions that did not interfere with Envfunction, and virus was surface immobilized for imaging via totalinternal reflection fluorescence microscopy (Munro, et al. BiophysicalJournal 104, 415A (2013)). smFRET trajectories were compiled intohistograms for the HIV-1_(JR-FL) Env trimer, either unliganded or afterpre-incubated for 30 min with 0.1 mg/ml PGT122, 35O22, or both PGT122and 35O22 prior to imaging. Resultant Env conformational landscapescould be deconvoluted into three gaussian distributions: a low-FRETpopulation that predominated for the prefusion mature unliganded state,and intermediate- and high-FRET populations, which predominated in thepresence of CD4 receptor and CD4-induced antibody (Munro, et al.Biophysical Journal 104, 415A (2013)). FIG. 7A, smFRET trajectories ofthe unliganded HIV-1_(JR-FL) Env trimer. Similar histograms wereobtained in the presence of PGT122 (FIG. 7B), 35O22 (FIG. 7C), and bothPGT122 and 35O22 (FIG. 7D).

FIGS. 8A-8C show antibody 35O22 and interface details. Despite thesubstantial immune evasion protecting the mature unliganded state fromhumoral recognition, after several years of infection, the human immunesystem does generate broadly neutralizing antibodies. One of these isthe 35O22 antibody, which neutralizes 62% of HIV-1 isolates at a medianIC₅₀ of 0.033 μg/ml. 35O22 binds parallel to the viral membrane, at agp120-gp41 epitope (FIG. 1B). FIG. 8A, 35O22 Fab, gp120 subunit, gp41subunit, and glycans are green. Complementary determining regions (CDRs)are labeled, and interactive HIV-1-Env residues highlighted in surfacerepresentation. At the membrane-distal surface of 35O22, an extendedframework 3 region (FW3) of the heavy chain (resulting from an insertionof 8 residues) interacts with strand β1 of the 7-stranded inner domainsandwich of gp120. The heavy chain-CDRs from extensive contacts with theN-linked glycan extending from residue 88_(gp120). In addition to glycancontacts, the CDR H3 of 35O22 interacts with the α9 helix of gp41. Helixα9 interactions are also made by the FW3 of the light chain (a completelist of contacts is provided in FIG. 28 ). Overall, 35O22 buries 1,105Å² solvent surface on gp120 (including 793 Å² with the Asn88_(gp120)glycan) and 594 Å2 solvent surface on gp41 (including 127 Å² with theAsn618_(gp41) glycan). Despite residue 625_(gp41) being part of theglycan sequon “NMT”, no glycan is observed; indeed, the side-chain amideof residue 625_(gp41) hydrogen bonds with the side-chain oxygen of Tyr32in the 35O22 heavy chain, and the presence of an N-linked glycan atresidue 625_(gp41) is difficult to reconcile with 35O22 recognition.FIG. 8B, 35O22 Fab shown in surface representation. FIG. 8C,2F_(o)-F_(c) at 1σ contour shown around glycan 88 of gp120. Antibody35O22 employs a novel mechanism of glycan-protein recognition, combininga protruding FW3 with CDR H1, H2 and H3 to form a “bowl” that holdsglycan. FW3 and CDR H3 provide the top edges of the bowl and interactwith the protein surface of gp120, whereas CDR H1 and H2 are recessedand hold/recognize glycan. This structural mechanism of recognitioncontrasts with the extended CDR 113-draping glycan observed with otherantibodies that penetrate the glycan shield such as PG9 and PGT128.

FIG. 9 shows a comparison of bound and unbound Fab conformations.Unbound and HIV-1-Env bound Fabs were superimposed, and ribbonrepresentations and rmsds are displayed here. (left), Regions whichshowed conformational changes are highlighted with black dotted lines. Acomplete list of contacts between PGT122 and BG505 SOSIP.664 is providedin FIG. 29 ).

FIGS. 10A and 10B show the principal component analysis of HIV-1 Env.Principle component analysis indicated each gp120-gp41 blade to form arectangle, with height of ˜100 Å, width of ˜65 Å, and thickness of ˜35Å. FIG. 10A, Minimum bounding box, generated by principle componentanalysis, in shown encasing the HIV-1-Env gp120-gp41 protomer; subunitsdisplayed in ribbon representation. As previously visualized (Walker, etal. Nature 477, 466-470 (2011)) the membrane-distal portion of therectangle is made up of the gp120-outer and -inner domains, with thecentral 7-stranded β-sandwich of the inner domain occupying thetrimer-distal, membrane-proximal portion of gp120. The rest of theectodomain trimer is now resolved: the membrane-proximal portion of therectangle is made up of gp41, with the membrane-distal portion of gp41closest to the molecular 3-fold axis occupied by a helix (whichcorresponds in register to the C-terminal portion of the postfusion HR1helix of gp41), and the rest of gp41 folding around N- andC-termini-strands of gp120, which extend over 20 Å toward the viralmembrane. FIG. 10B, Different views of trimeric protomer association.

FIGS. 11A and 11B show the conformational changes between gp120prefusion and CD4-bound state. FIG. 11A, gp120 from BG505 (positions31-511 of SEQ ID NO: 2) is shown in cartoon representation in prefusion(dark grey) and CD4-bound (light grey, PDB ID 3JWD) conformation. V1V2(PDB ID 3U2S) has been modeled onto the CD4-bound conformation.Secondary structure changes from prefusion to CD4-bound conformation areshown with cylinders representing α-helix and arrows β-strands.Disordered residues are indicated by “X”. Residues that move more than 3Å between the prefusion and the CD4-bound conformations are shown withgrey shadows. FIG. 11B, Details of conformational changes between theprefusion and the CD4-bound conformation of gp120 (shown in cartoon):regions highlighted cover layer 1 with α0 changes, layer 2 with α1changes and β20-21 rearrangements. All atoms rmsd for the followingregion are: for residues 54-74 in gp120, rmsd=4.759; for residues 98-117in gp120, rmsd=0.497; for residues 424-436 gp120, rmsd=3.196. FIG. 11Ais produced in color as extended data FIG. 4 a in Pancera et al.,Nature, 514(7523, 455-461, 2014, extended data FIG. 4 a of Pancera etal. is incorporated by reference herein.

FIGS. 12A-12C show the relative protein surface occlusion by glycans.The solvent-accessible protein surface is shown in red and N-linkedglycans are shown in green. Calculations of the percentage coverage ofthe protein surface were determined for the four trimer models based ontwo probe sizes of 1.4 Å (solvent radius) and 10.0 Å (the estimatedsteric footprint of an antibody combining region). FIG. 12A, EstimatedMan-9 glycan coverage. FIG. 12B, Estimated Man-5 glycan coverage. FIG.12C, Visualization of Man-9 N-linked glycan coverage for two proberadii. Surface area calculations were carried out according to Kong etal. (Kong, et al. Journal of molecular biology 403, 131-147 (2010)) andimages were generated using Grasp v1.3 (Nicholls, et al. Proteins 11,281-296 (1991)). The PDB IDs associated with the glycosylated modelsare: 4TVP (HIV-1), 2YP7 (Flu) and 4JHW (RSV).

FIGS. 13A and 13B show the prevalence of neutralizing responsesidentified serologically from cohorts from (FIG. 13A) years 2-3 and(FIG. 13B) years 5+ post infection. For each serum, the predictedneutralization prevalence for each of 12 antibody specificities is shownbased on neutralization of 21 diverse HIV-1 strains. Serumneutralization on 21-strain virus panel is shown in FIG. 30 .

FIG. 14 is a table providing data collection and refinement statisticsfor the BG505.SOSIP.664-PGT122-35O22 protein complex structure.

FIG. 15 is a table listing the modeling parameters for gp120 and gp41rearrangements. To provide reference frames for the various prefusionconformational states, we extracted Env component of SOSIP bound byVRC-PG04 (Lyumkis, et al. Science 342, 1484-1490 (2013)) and by VRC03(Bartesaghi, et al. Nature structural & molecular biology 20, 1352-1357(2013)) and the resultant maps with the CD4-bound conformation trimericBAL (Tran, et al. PLoS pathogens 8, e1002797 (2012)). Once maps werealigned, gp120 and gp41 models were fit to each of the maps as definedin the table in black text after “gp120” and “gp41”. In addition torigid-body fits of crystal structures, specific regions of gp120 andgp41 were modeled and are defined in the table in red text afterdifferent portions of gp120 and gp41 relative to the prefusion matureclosed conformation.

The prefusion mature closed conformation of gp120 and gp41 wasestablished from the crystal structure presented in Example 1 and wasfit without modification to EMDB-5779 with density from PGV04 fabscomputationally removed. The prefusion partially open intermediateconformation was modeled by a rigid body fitting of gp120 to EMDB-2484with density from VRC03 Fabs computationally removed. α7 of gp41 wasextended into the unoccupied density at the N-terminus of the helixusing the mature closed structure as a starting model. The prefusionreceptor-bound intermediate was modeled by fitting the CD4-bound gp120core crystal structure (PDB ID 3JWD) to the CD4-bound EMDB-5455 map. V3of the crystal structure (PDB ID 3H11) was aligned to the core and theV1V2 crystal structure (PDB ID 3U4E) was fit to the remaining density.α7 of gp41 was extended through an alignment with crystal structures ofpostfusion gp41 (PDB IDs 2X7R, 2EZO). Postfusion gp120 is in the sameconformation as the prefusion receptor-bound intermediate and thepostfusion gp41 structure was derived from an alignment of SIV and HIVpostfusion crystal structures (PDB IDs 2X7R, 2EZO).

FIGS. 16A and 16B are tables showing binding parameters of the 35O22,PGT151, and PGT145 antibodies to trimeric BG505 SOSIP.664. *NBD: Nobinding detected, (double dagger) SE: Standard error calculated fromglobal fit of 6 independent injections. FIG. 16A: #Levels of capturedtrimer varied between 400-500 RU for the CD4 Ig and 2G12 captures,whereas ˜1500 RUs of trimer (+sCD4) was captures in the 17b captureformat. FIG. 16B: SE: Standard error calculated from global fit of 6independent injections; SD: Standard deviation of trimer capture from 6independent injection; Normalized: (R-/Level of trimer capture)*100.

FIG. 17 is a table showing the atomic-level structures for HIV-1-Envregions determined in complex with HIV-1-neutralizing antibodies.Neutralizing antibodies generally recognize the prefusion conformationof HIV-1 Env. Thus structures highlighted here display a cumulative sumtotal of prefusion HIV-1-Env structural information. Env residues arenumbered according to standard HXB2 numbering (from PDBs). Onestructure, for antibody D5, is in the postfusion gp41 conformation, andis thus not included in the sum total. Other structures for PDB 4CC8,4NCO, and 3J5M, do not define sequence register, and are also notincluded in the sum total.

FIGS. 18A-18C show biolayer interferometry binding profiles ofmonoclonal antibodies to BG505 SOSIP.664. Octet Biosensorgrams of BG505SOSIP.664 (FIG. 18A) or BG505 gp120 (FIG. 18B) binding to humanmonoclonal IgGs. Human monoclonal antibodies were loaded onto AMC probesand association with gp140 or gp120 proteins (at 50 μM concentration)were allowed to proceed for 300s, followed by dissociation for 300s withthe responses measured in nm using an Octet Red 384 machine. Allexperiments were carried out at 30° C. in PBS buffer (pH 7.4)supplemented with 1% BSA to minimize non-specific binding. The dottedline indicates the beginning of the dissociation phase and the maximalspecific binding after 300s reported in the table shown in FIG. 18C (−no binding, + from background to 0.175 RU, ++ 0.175 RU to 0.35 RU and+++ from 0.35 RU to 0.8 RU). BG505gp120 did not contain the T332Nmutation (no glycan at that position). The antigenicity of the BG505SOSIP.664 protein varied depending on the assay done. Thus, usingsurface plasmon resonance, no CD41 antibodies binding was detected whilesome binding could be observed using biolayer interferometry.

FIGS. 19A-19B are a set of ribbon diagrams showing modeling of gp41residues 548-568. At low contour, suggestive density is observed thatmight correspond to the connection between α6 and α7 helices. Toinvestigate the degree to which a model for this region might bedefined, two different models for this region were built and refined, asshown in FIG. 19A, with electron density shown for 2F₀-F_(c) density at1σ contour. The location of the I/P mutation at 559 is indicated. FIG.19B, Superimposed models shown in perpendicular orientations.

FIGS. 20A-20C illustrate the gp120-gp41 and gp41-gp41 interfaces. FIG.20A, Cartoon representation of gp120 and gp41. Region of gp120 thatinteracts with gp41 is shown in surface representation and region ofgp41 that interacts with gp120 is shown in semitransparent surface. FIG.20B, gp41-trimer interfaces as viewed from the viral membrane in ribbonand surface representation (90° rotation from FIG. 2C). FIG. 20C,Residues that have been shown by mutagenesis (Helseth et al., J Virol,65, 2119-2123 (1991); Thali et al., J Virol, 66, 5516-5524 (1992); Cao,J. et al. J Virol, 67, 2747-2755 (1993); Leavitt et al., J Virol, 77,560-570 (2003); Yang et al., Virology 313, 117-125 (2003); Sen et al.,Biochemistry 47, 7788-7795 (2008); Wang et al., J Biol Chem 283,32644-32649 (2008)) to be important for gp120/gp41 association areunderlined on sequence and residues that are shown to interact betweengp120 and gp41 from the crystal structure are indicated in red from thesame protomer and in orange if between two protomers. The sequence ofHIV-1 Env ectodomdina from BG505 (positions 31-664 of SEQ ID NO: 2 isshown). Sites of N-linked glycosylation are shown in green.

FIG. 21 illustrates HIV-SIV postfusion chimera. Sequences of HIV-1 gp41prefusion, postfusion (HIVpost, PDB ID: 2X7R) and SIV postfusion(SIVpost, PDB ID: 2EZO) are aligned with secondary structure indicated.Residues that were used to make the postfusion HIV-1/SIV chimera used inFIG. 3 are shown in red.

FIGS. 22A and 22B illustrate the fusion-intermediate entry inhibitors ofHIV-1 envelope. FIG. 22A, The binding residues of representativefusion-intermediate targeting entry inhibitors or antibodies were mappedonto the structure of pre-fusion envelope (Lawless et al., Biochemistry35, 13697-13708 (1996); Chen et al., J Virol, 69, 3771-3777 (1995); Rootet al., Science 291, 884-888 (2001)). Upper panels, ribbonrepresentation of pre-fusion envelope protomer A, at two orientations,with the binding residues of the fusion-intermediate inhibitors 5-helix,T20, and monoclonal antibody D5 indicated. Lower panels, surfacerepresentation of the pre-fusion envelope trimer, with thefusion-intermediate binding residues mapped onto the surfaces of allprotomers. gp120 is colored lighter gray and gp41 is colored darkergrey. Binding residues of fusion-intermediate inhibitors 5-helix, T20,and monoclonal antibody D5 (Luftig et al., Nature structural & molecularbiology 13, 740-747 (2006)) are indicated. FIG. 22B, Fusion-intermediateentry inhibitors T20, 5-helix, and D5 Fab docked onto a model offusion-intermediate gp41.

FIG. 23 shows a set of graphs showing the effect of CD4 and CD4/17b onbinding of antibodies 35O22 and PGT151 to BG505 SOSIP.664. The structureof a near-native prefusion state of HIV-1 provides a critical additionto the pantheon of HIV-1 Env structures with atomic-level detail.Moreover, antibodies 35O22 and PGT151, which bind specifically to thetrimeric prefusion conformation of gp41, provide new tools by which toassess the conformational state of gp41 (Blattner et al., Immunity 40,669-680 (2014); Falkowska et al., Immunity 40, 657-668 (2014). Thebinding of antibodies 35O22 and PGT151 to BG505 SOSIP.664 trimer wastested in the presence of the CD4 receptor and the 17b antibody (Thaliet al., J Virol, 67, 3978-3988 (1993) (a co-receptor surrogate whichrecognizes a bridging sheet epitope that overlaps the site ofco-receptor recognition). In the case of antibody 35O22, CD4 binding tothe BG505 SOSIP.664 trimer impacted the kinetics, affinity andstoichiometry of binding. 35O22 bound to BG505 SOSIP.664 with an8.4-fold reduced affinity, primarily contributed by an increased rate ofdissociation. The overall binding level (Rmax) normalized to the averagelevel of trimer captured (see also FIG. 16 ) was lower suggestingsubstoichiometric binding. Capturing the trimer on a CD4-Ig surfacereduced normalized R_(max) for PGT151 compared to the 2G12 captureformat, suggesting reduced stoichiometry for PGT151 binding to trimerpre-bound with CD4, although kinetics and affinity of interaction weresimilar. A BG505 SOSIP.664 trimer+sCD4 complex captured onto a 17bsurface bound 35O22 but showed no detectable binding to PGT151.

FIGS. 24A and 24B illustrate the postfusion binding pocket of gp41 claspresidues Trp628 and Trp631 is targeted by neutralizing antibodies. FIG.24A, Shown are ribbon representations of gp41 5-helix protein (Root, etal 2001) (left) docked with an additional C-heptad repeat (CHR) helix(middle panel,) or with a representative neutralizing antibody, D5, thattargets this site (Luftig et al., Nature structural & molecular biology13, 740-747 (2006); Gustchina et al., PLoS pathogens 6, e1001182 (2010);Sabin et al, PLoS pathogens 6, e1001195 (2010)) (right panel). Residuesof the prefusion clasp, W628 and W631, that are part of CHR areindicated. N-heptad repeat (NHR) helices and CHR helices are indicated.FIG. 24B, Surface representation of 5-helix protein (same orientationand coloring as in FIG. 24A) is shown with the footprints of gp41 claspresidues W628 and W631 (middle panel) and antibody D5 (right panel).

FIGS. 25A-25E are a set of tables showing binding and contact parametersfor the gp120-gp41 interface in the HIV-1 viral spike. D: Disulfidebond, H: Hydrogen bond, S: Salt bridge. ASA: Accessible Surface Area,Å², BSA: Buried Surface Area, Å², ΔiG: Solvation energy effect,kcal/mol,

: Buried area percentage, one bar per 10%.

FIG. 26 is a table and a set of ribbon diagrams illustrating structuresthat are related to the gp120/gp41 structure in the prefusion matureclosed conformation of the HIV-1 Env ectodomain trimer.

FIGS. 27A and 27B illustrate qualitative recognition of HIV-1 envelopeby diverse antibodies is shown for five conformational states. Lightgrey bars indicate anticipated recognition, dark grey bars norecognition, and absence of a bar indicates that recognition isundefined. HIV-1 recognition is from cited references (Luftig et al,Nature structural & molecular biology 13, 740-747 (2006); Blattner etal., Immunity 40, 669-680 (2014); Gustchina et al, PLoS pathogens 6,e1001182 (2010); Sabin et al, PLoS pathogens 6, e1001195 (2010); Liao etal., Nature 496, 469-476 (2013); Zhou et al., Science 329, 811-817(2010); Scheid et al., Science 333, 1633-1637 (2011); Sanders et al.,PLoS pathogens 9, e1003618 (2013); Yasmeen et al., Retrovirology 11, 41(2014); Lyumkis et al., Science 342, 1484-1490 (2013); Ringe et al.,PNAS USA 110, 18256-18261 (2013); Julien et al., Science 342, 1477-1483(2013); Walker et al., Nature 477, 466-470 (2011); Stanfield et al.,Human antibodies 14, 73-80 (2005); Pancera et al., PNAS USA 107,1166-1171 (2010); Huang et al., Science 310, 1025-1028 (2005); Huang etal., Science 317, 1930-1934 (2007); Rizzuto et al., Science 280,1949-1953. (1998); Guan et al., PNAS USA 110, E69-78 (2013); Gomy etal., Virology 267, 220-228 (2000); Yuan et al., AIDS research and humanretroviruses 25, 319-328 (2009); Moore et al., J Virol, 80, 2515-2528(2006); Miller et al., PNAS USA 102, 14759-14764 (2005); Chen et al., JVirol, 88, 1249-1258 (2014); Chakrabarti et al., AIDS research and humanretroviruses 27, 877-887 (2011); Frey et al., PNAS USA 105, 3739-3744(2008); Nicely et al., Nature structural & molecular biology 17,1492-1494 (2010); Huang et al., Nature 491, 406-412 (2012)) or fromantibodies 35O22 and PGT151 recogition of HIV-1 Env as shown herein (seeFIG. 16 ).

FIGS. 28A-28D and 29A-29D show a set of tables listing binding andcontact parameters for the interaction of the 35O22 heavy and lightchains variable regions with the trimeric HIV-1 Env ectodomain. D:Disulfide bond, H: Hydrogen bond, S: Salt bridge. ASA: AccessibleSurface Area, Å², BSA: Buried Surface Area, Å², ΔiG: Solvation energyeffect, kcal/mol,

: Buried area percentage, one bar per 10%.

FIGS. 30A and 30B are a set of tables listing serum neutralizationresults from a 21-strain virus panel.

FIG. 31 is a schematic diagram illustrating structural implementation ofHIV-1 evasion.

FIGS. 32A-32D are a set of ribbon diagrams and graphs illustrating thatthe unliganded HIV-1 Env trimer is structurally compatible with epitopesof broadly neutralizing but not ineffective antibodies. FIG. 32A,Superposition of unliganded and antibody bound HIV-1 Env structures.(Left) The unliganded gp120 core monomer is shown in ribbonsrepresentation, with regions of less (or greater) than 2 Å RMSD uponantibody binding indicated, and representative antibody-bound structuresin black. (Middle and right) Unliganded and antibody-bound HIV-1 Envtrimers. (In the right panel, antibodies PGT122 and 35O22 are shown ingray semitransparent surfaces, and the rear protomer has been removedfor clarity). FIG. 32B, Breadth-potency of broadly neutralizing andineffective antibodies on a diverse 170 HIV-1 isolate panel. FIG. 32C,Unliganded Env structure is displayed as a Ca-ribbon, with antibodyepitope residues light grey (structurally compatible) or dark grey(incompatible). RMSD (solid fill) and volume overlap (striped fill) withthe respective antibody-Env complexes are shown as a bar graph, with twolinear scales split at the RMSD and antibody-antigen volume overlapcutoffs of 2 Å and 500 Å³, respectively; bars below the respectivecutoffs. Antibody labels are dark grey if incompatible, and gray if notpresent in the structure. FIG. 32D, Structural compatibility versusbreadth. Volume overlap (left), RMSD (middle) and Antigenic StructuralCompatibility score (ASC) (right) are graphed versus antibody breadth ona diverse 170 HIV-1 isolate panel. P-values for Spearman correlationsprovided.

FIGS. 33A-33F are a set of diagrams and graphs illustrating theantigenicity and conformational fixation of unliganded HIV-1 Env. FIG.33A, BG505 SOSIP.664 structural compatibility versus bindingantigenicity, in the absence (left) and presence (right) of CD4; averagebinding of each antibody is provided, and ineffective antibodies labeled(CD4bs:CD4-binding site, CD41: CD4-induced, non: non-neutralizing, V3:V3-loop directed). FIG. 33B, Conformationally stabilized BG505 SOSIP.664variants. The central image depicts the unliganded BG505 SOSIP.664 HIV-1Env trimer, with two protomers shown in cartoon representation. Insets:atomic-level details. *Residue 559 is disordered in the unligandedstructure. FIG. 33C, Binding antigenicity of BG505 SOSIP.664 andalterations to BG505 SOSIP.664 that stabilize the unliganded closedstate. Heat map shows binding of BG505 SOSIP.664 and variants to a panelof antibodies. A433P was temporally less stable than 201C-433C (ExtendedData FIG. 6 b ) FIG. 33D, Structure of BG505 SOSIP.664 201C-433C iscompatible with binding antigenicity, in both the absence (left) andpresence (right) of CD4, with antibodies and average binding of broadlyneutralizing and ineffective antibodies as in FIG. 33A. FIG. 33E,Atomic-level models of residues 201 and 433 in unliganded prefusionclosed and CD4-bound conformations. Ribbon representation of the twostructures are shown with bridging sheet shown in dark grey, residues201 and 433 highlighted and Cα distance between the two indicated.Positions of the variable loops are shown. (Monomeric CD4-boundconformation from PDB ID 3JWD, 3U4E and 2B4C.) FIG. 33F, Differentialscanning calorimetry.

FIGS. 34A-34F are a set of diagrams and graphs illustrating thatunliganded HIV-1 Env binds a single CD4 without the typical antigenichallmarks of CD4 triggering. FIG. 34A, Binding of soluble CD4 toSOSIP.664 and mutants that stabilize the prefusion mature closed statemeasured by SPR (CD4 on chip). FIG. 34B, Kinetics of CD4 activation bySPR measured by binding to 17b, which recognizes a bridging sheetepitope (17b on chip; left) and to 3074, which recognizes a V3 epitope(3074 on chip; right). FIG. 34C, Sedimentation equilibrium analyticalultracentrifugation measurements of BG505 SOSIP.664 and 201C-433Cvariant in presence of excess soluble CD4. FIG. 34D, Butterfly plotsdisplay the HDX profiles of BG505 SOSIP.664 DS variant (left) and DSvariant with CD4 (right). The percent exchange is plotted for eachpeptide; raw difference plots are shown below. Qualitative changes byHDX are displayed on one lobe of the unliganded trimer with regionsbecoming more ordered and disordered. FIG. 34E, smFRET of JR-FL virionswith and without 201C-433C substitution. Population FRET histograms areeach paired with transition density plots, which display the relativedensity of observed transitions. FIG. 34F, HIV-1 entry mechanism withconformation-blocking mutations, antigenicity, and interactions withfunctional ligands. The results reveal a new mechanistic state, which ischaracterized by the binding of a single molecule of CD4, no bridgingsheet formation and reduced V3 loop exposure.

FIGS. 35A and 35B are a set of graphs illustrating properties ofconformationally fixed HIV-1 Env trimeric immunogens. FIG. 35A, Physicalstability of trimeric BG505 SOSIP.664 201C-433C as determined by thequaternary specific antibody VRC26.09 after 60 minutes of incubation atextremes of temperature, pH, or ten freeze-thaw cycles. FIG. 35B,Virus-like particle (VLP) antigenicity. Strain JR-FL was modified withE168K to allow binding of V1V2-directed broadly neutralizing antibodies;strain BG505 was modified with T332N to allow binding of 2G12 antibody.While broadly neutralizing antibody binding is maintained between parentand 201C-433C VLPs, the 201C-433C variant shows reduced ineffectiveantibody binding, especially in the presence of CD4. Ineffectiveantibodies labeled (CD4bs: CD4-binding site, CD41: CD4-induced, V3:V3-loop directed) FIGS. 36A-36D are a set of graphs and tablesillustrating the characterization of purified BG505 SOSIP.664 andselected variants. FIG. 36A, Properties of purified gp140 proteins. *thepercentage of trimers was obtained by measuring area under the curve ofthe gel filtration profile of the various peaks representing aggregates,gp140 trimer, dimer and monomer. FIG. 36B, gel filtration profiles onSuperdex 200 with indicated line showing a second round of purificationwhen performed. Dotted lines show the fractions selected for analyses.FIG. 36C, 2D class averages from a reference-free classification ofnegative stained EM data for each protein. Box size=28 nm. FIG. 36D,447-52D negative selection. Trimeric peak of BG505 SOSIP.664 purifiedover gel filtration is used as starting point. Protein is passed over a447-52D affinity column and flow through collected as well as eluatefrom the 447-52D column (with 3 M MgCl2). SDS-page under reducing (R)and non-reducing conditions (NR) is shown with protein before 447-52negative selection, protein after 447-52D negative selection and eluatefrom the 447-52D affinity column. A higher molecular weight band can beremoved by 447-52D negative selection. SPR measurement of the same threefractions, before, after and eluate binding to 2G12, 447-52D and CD4.The portion of the protein that binds to 447-52D can be removed bynegative selection while maintaining CD4 and 2G12 binding. 80% of theprotein is recovered after 447-52D negative selection.

FIGS. 37A-37E are a set of graphs illustrating the antigenicity ofpurified BG505 SOSIP.664 and selected variants by MSD and ELISA. FIG.37A, Antigenicity of BG505 SOSIP.664 and mutants by MSD-ECLIA. Plotsshow binding of neutralizing, non- or weakly neutralizing antibodies(dark grey) and antibodies in presence of CD4 to BG505 SOSIP.664 andmutants. FIG. 37B, Temporal stability of BG505 SOSIP.664, A433P and201C-433C. FIG. 37C, Comparison of antigenicity of BG505 SOSIP.664 and201C-433C in absence and presence of soluble, 2-domain CD4. FIG. 37D,Antigenicity of BG505 SOSIP.664 and 201C-433C assessed by ELISA. FIG.37E, Pair wise comparison of antigenicity data obtained by MSD-ECLIA andELISA.

FIGS. 38A-38D are a set of graphs and tables illustrating theantigenicity of purified BG505 SOSIP.664 and 201C-433C by SPR and BLI.Binding of BG505 SOSIP.664 and 201C-433C measured by FIG. 38A, SurfacePlasmon Resonance and FIG. 38B, Biolayer Interferometry. FIG. 38C,Affinities of BG505 SOSIP.664 and 201C-433C to neutralizing andnon-neutralizing antibodies by SPR and Biolayer interferometry. FIG.38D, Pairwise comparison of antigenicity data by 4 methods-MSD, ELISA,BLI and SPR. The P values were corrected for false discovery rate formultiple comparisons.

FIGS. 39A-39D are a set of graphs illustrating the characterization ofBG505 SOSIP.664 and 201C-433C binding to CD4-induced epitopes in absenceand presence of CD4 by FIG. 39A, MSD-ECLIA FIG. 39B, SPR with antibodiescaptured on an anti-Fc surface and trimer as analyte, SOSIP is shown insolid line and 201C-433C in dashed line, trimer without CD4 is dark greyand trimer with CD4 light grey. FIG. 39C and FIG. 39D, ELISA at 0.5μg/ml and 2 μg/ml trimer captured on D7324-coated plates, respectively.

FIGS. 40A-40E are a set of graphs illustrating the CD4-inducedactivation of HIV-1 Env. FIG. 40A, SPR analysis of (top panel)co-receptor site exposure and (bottom panel) HR2-site exposure upon CD4activation. FIG. 40B, CD4-induced binding of SOSIP and P313W mutant to17b. Trimer samples at concentrations from 40 nM to 2.5 nM in 2-folddilutions were combined with 200 nM sCD4 and injected on a 200RU 17b IgGsurface. FIG. 40C, SPR single-cycle kinetics analysis of 17b Fab bindingto soluble trimers activated with sCD4. Under conditions of constant 50nM sCD4 flow, 17b Fab at was injected incrementally in 2-fold dilutionsfrom 25 nM to 1.5 nM on trimer captured on a 2G12 chip. A433P and201C-433C were further subjected to 17b Fab concentrations ranging from500 nM to 31.25 nM (insets). FIG. 40D, Time-dependent increase inexposure of V3 epitope (3074 detection) and bridging sheet (17bdetection) in SOSIP trimers on incubation with sCD4. FIG. 40E, Abilityof HIV-1 BG505 and mutants to enter CD4+CCR5+ cells.

FIG. 41 is a set of graphs illustrating the analysis of stoichiometry ofCD4-binding to BG505SOSIP.664.332N or the DS variant, BG505SOSIP201C-433C. Masses were determined for each molecule by MALDI-TOF massspectrometry, as follows: BG505.SOSIP.664 ((109,994±11 Da)×3=329,982±33Da), BG505.SOSIP.664 201C-433C ((109,006±10 Da)×3=327,018±30 Da), andCD4 d1d2 (20,329±2 Da). For the BG505 SOSIP molecules, monomer masseswere determined, but trimeric masses were used in the analyticalultracentrifugation fitting. Four fittings are shown for eachexperiment, with fits calculated for 0, 1, 2, or 3 bound CD4 molecules,respectively. For BG505SOSIP.664.N332, the residual errors switchdirection between 1:2 and 1:3 stoichiometries, suggesting 2-3 CD4molecules bound. For the DS mutant, BG505SOSIP 201C-433C, the residualerrors fit best at 1:1 stoichiometry and fail to fit higher numbers ofbound CD4 molecules, indicating 1 CD4 molecule bound per DS trimer.

FIGS. 42A-42G, 43 and 44 are a set of tables showing antigeniccharacteristics of recombinant HIV-1 Env ectodomain trimers (FIGS.42A-42G, and 44 ), protein nanoparticles including recombinant HIV-1 Envectodomain trimers (FIG. 43 ). The binding activity of the recombinantHIV-1 Env trimers or protein nanoparticles including recombinant HIV-1Env ectodomain trimers was compared to that of the HIV-1 Env BG505SOSIP.664 construct by ELISA assay. “+++” indicates binding within 75%of HIV-1 Env BG505 SOSIP.664, “++” indicates binding between 50-75% ofHIV-1 Env BG505 SOSIP.664, “+” indicates binding within 25-50% of HIV-1Env BG505 SOSIP.664, and “−” indicates binding within 0-25% of HIV-1 EnvBG505 SOSIP.664.

FIGS. 45 and 46 are schematic diagrams illustrating the construction ofchimeric HIV-1 Env ectodomain trimers including gp120 N- and C-terminalsequences from a first HIV-1 strain (shown in lighter grey) with theremainder of gp120 from a second HIV-1 strain (shown in darker grey).

FIG. 47 is a ribbon diagram showing a single protomer of the HIV-1 Envectodomain in a mature prefusion closed conformation, with certainstructural elements and residues indicated. The β-4 and β-3 strands atthe N-terminal region of gp120 and the $26 and 325 strands and α5 helixat the C-terminal region of gp120 indicated. Many of the residues ofgp120 that interface with gp41 (residues 46-54, 70-75, 84-89, 99, 102,106, 107, 114, 215, 220-224, 226, 244, 471-473, 476-477) are present.

FIG. 48 shows a set of coommassie stained SDS-PAGE gels and elutionprofile graphs illustrating purification chimeric HIV-1 Env ectodomaintrimers including sequences of the BG505 HIV-1 strain and one of the3301, ZM53, CAP256-SU, and 25925 HIV-1 strains.

FIG. 49 is a set of graphs illustrating the antigenicity of chimericHIV-1 Env trimers including the ZM53_BG505-NCgp120_gp41.SOSIP (SEQ IDNO: 386), 25925-2.22_BG505-NCgp120_gp41.SOSIP (SEQ ID NO: 383), and3301_V1_C24_BG505-NCgp120+gp41.SOSIP (SEQ ID NO: 384) constructs.

FIG. 50 shows a coommassie blue stained polyacrylamide gel and a graphillustrating expression, purification, and antigenicity of a chimericHIV-1 Env trimers including a gp120 sequence from the BG505 strain, agp41 sequence from the CAP45 strain, and the SOSIP mutations (SEQ ID NO:772).

FIG. 51 is a set of graphs illustrating that the neutralization profileof the indicated antibodies for the native DU156 virus correlates withthe antigenic profile of a recombinant HIV-1 Env ectodomain trimerstabilized in the prefusion mature closed conformation as disclosedherein.

FIG. 52 shows a set of electron micrograph images indicating thatpurified CNE58-strandC, CAP256-SU, and 3301_V1_C24_bg505 chimeric HIV-1Env ectodomains attain trimeric closed configuration.

FIGS. 53A-53E are a set of tables illustrating the antigeniccharacteristics of the indicated recombinant HIV-1 Env ectodomaintrimers. A “i” following the name of recombinant Env ectodomainindicates that the assayed HIV-1 Env ectodomain is a chimeric HIV-1 Envectodomain including gp120 residues 31-45 and 478-507, and gp41 residues512-664 from the BG505 strain with SOSIP substitutions, and a disulfidebetween residues 201-433, with the remainder of the gp120 sequence froma second HIV-1 strain. A “2” following the name of recombinant Envectodomain indicates that the assayed HIV-1 Env ectodomain is a chimericHIV-1 Env ectodomain including gp120 residues 31-45, 478-507, andInterface Residue Set A (see Example 5), and gp41 residues 512-664 fromthe BG505 strain with SOSIP substitutions, and a disulfide betweenresidues 201-433, with the remainder of the gp120 sequence from a secondHIV-1 strain. A “3” following the name of recombinant Env ectodomainindicates that assayed HIV-1 Env ectodomain is a chimeric single chainHIV-1 Env ectodomain including gp120 residues 31-45 and 478-507, andgp41 residues 512-664 from the BG505 strain with SOSIP substitutions,and a disulfide between residues 201-433, with the remainder of thegp120 sequence from a second HIV-1 strain, and peptide linker in placeof the protease cleavage site between gp120 and gp41. A “4” followingthe name of recombinant Env ectodomain indicates that the assayed HIV-1Env ectodomain includes the SOSIP substitutions is a chimeric proteinincluding gp120 residues 31-34 from the BG505 strain with SOSIPsubstitutions, and a disulfide between residues 201-433, with theremainder of the gp120 and gp41 sequences from a second HIV-1 strain.

FIGS. 54A and 54B are a set of graphs illustrating results from ELISAassays showing that the BG505.SOSIP.664.201C-433C HIV-1 Env ectodomaintrimer induces production of HIV-1 Env trimer specific and V3-peptidespecific antibodies in rabbits (FIG. 54A) and guinea pigs (FIG. 54B).

FIGS. 55A-55C are a table and a set of graphs showing results from HIV-1neutralization assays using sera collected from rabbits or guinea pigsimmunized with the BG505.SOSIP.664 HIV-1 Env ectodomain trimer or theBG505.SOSIP.664.201C-433C “DS” HIV-1 Env ectodomain trimer. Eachimmunogen elicited comparable autologous virus (BG505.W6M.C2.T332N) andV3 directed tier 1 virus (MW965.26) neutralizing activity as measured byIC₅₀. A summary of the results for autologous virus and V3 directed tier1 virus is provided for guinea pig week 18 sera (FIG. 55B) and rabbitweek 18 sera (FIG. 55C).

FIG. 56 is a table listing antigenic characteristics of chimeric HIV-1Env ectodomains linked to a transmembrane domain as expressed on surfaceof cells.

FIG. 57 is a graph and a sequence alignment showing that the inferredancestor and intermediates of V1V2-directed bNAbs neutralize a commonset of HIV-1 isolates. “Neutralized” represents the number of HIV-1strains with IC₅₀ of less than 50 μg/ml, and “Total” indicates thenumber of HIV-1 strains tested; IC₅₀ for select strains is indicated bya colored dot. Nomenclature of the revertants is as follows: unmutatedcommon ancestor (UCA), reverted V-gene, mature CDR3 (gHgL), earlyintermediate from next-generation sequencing (Il). The sequence of theV1V2 domain (positions 129-196) of the BG505 (SEQ ID NO: 2), CAP256.SU(SEQ ID NO: 51), BB201.B42 (SEQ ID NO: 81), KER2018.11 (SEQ ID NO: 107),CH070.1 (SEQ ID NO: 174), ZM233.6 (SEQ ID NO: 745), Q23.17 (SEQ ID NO:746), A244 (SEQ ID NO: 747), T250-4 (SEQ ID NO: 2114), and WITO.33 (SEQID NO: 748) strains of HIV-1 is shown.

FIGS. 58A-58C illustrate the design and antigenicity of HIV-1 Envectodomain trimer immunogens stabilized in the prefusion mature closedconformation that include a chimeric V1V2 domain sequence. (FIG. 58A)Design of chimeric V1V2 DS-SOSIP.664 trimers. Residues 126-196 ofstrains found to preferentially interact with germline-revertedV1V2-directed antibodies were transferred to the corresponding region ofBG505 DS-SOSIP.664, with D368R mutation. (FIG. 58B) Gel filtration andnegative stain EM (2D class averages) of BG505SOSIP.664.DS.368R.CAP256-SU, a representative chimera. (FIG. 58C)Binding of chimeric DS-SOSIP.664s and neutralization of correspondingpseudoviruses by ancestors, intermediates, and mature V1V2-directedbNAbs. Antibodies are listed in the left column, and HIV-1 strains arelisted across the top; results are tabulated in double cells, with theleft cell showing binding and the right cell showing neutralization.Immunogens contain the 201C-433C disulfide mutation for stabilizationand also contain a D368R CD4 binding site knock out mutation to preventthe trimer from opening in vivo.

FIG. 59 is a table illustrating the antigenic characteristics of theindicated recombinant HIV-1 Env ectodomain trimers, which are chimericHIV-1 Env ectodomain stabilized in the prefusion mature closedconformation and include BG505 and JRFL sequences.

FIG. 60 is a set of graphs showing binding to VRC20gHgL and VRC01 gHgLunmutated common ancestor (UCA) antibodies, as well as the indicatedneutralizing antibodies by a chimeric HIV-1 Env ectodomain trimerincluding a BG505 “platform” and 426c gp120 residues with mutation ofthe glycan sequons at positions 276, 460 and 463 and the “DS”substitutions (201C/433C).

SEQUENCES

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and amino acids, as defined in 37C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown,but the complementary strand is understood as included by any referenceto the displayed strand. The Sequence Listing is submitted as an XMLfile in the form of the file named “4239-93056-13 Sequence Listing.xml”(4,035,499 bytes), which was created on Sep. 20, 2022, which isincorporated by reference herein.

Table 13 in Example 15 provides a list of sequences and additionalinformation concerning the sequences.

STRUCTURAL COORDINATES

The atomic coordinates of an asymmetric unit of the crystal structure ofa trimeric HIV-1 Env ectodomain (BG505.SOSIP.664) bound to PGT122 and35O22 Fabs in the prefusion mature closed conformation (as described inExample 1) are recited in Table 1 submitted as an ASCII text named“Table_1.txt” (˜2 MB, created on Aug. 7, 2014) in U.S. ProvisionalApplication No. 62/046,059, filed Sep. 4, 2014, and have been depositedwith the Protein Data Bank as Acc. No. 4TVP. Table 1 submitted in U.S.Provisional Application No. 62/046,059, and Protein Data Bank Ace. No.4TVP, are incorporated by reference herein.

The atomic coordinates of the crystal structure of an HIV-1 Envectodomain trimer provided in Table 1, without the PGT122 and 35O22Fabs, are recited in Table 2 submitted as an ASCII text named“Table_2.txt” (˜2 MB, created on Aug. 7, 2014) in U.S. ProvisionalApplication No. 62/046,059, filed Sep. 4, 2014. Table 2 provided in U.S.Provisional Application No. 62/046,059 is incorporated by referenceherein.

The atomic coordinates of an asymmetric unit of the crystal structure ofan unliganded trimeric HIV-1 Env ectodomain in the prefusion matureclosed conformation (as described in Example 2) are recited in Table 3submitted as an ASCII text named “Table_3.txt” (˜0.7 MB, created on Aug.7, 2014) in U.S. Provisional Application No. 62/046,059, filed Sep. 4,2014, and have been deposited with the Protein Data Bank as Acc. No.47MJ. Table 3 submitted in U.S. Provisional Application No. 62/046,059,and Protein Data Bank Acc. No. 47MJ, are incorporated by referenceherein.

The atomic coordinates of the crystal structure of an unligandedtrimeric HIV-1 Env ectodomain in the prefusion mature closedconformation (as described in Example 2) are recited in Table 4submitted as an ASCII text named “Table_4.txt” (˜2 MB, created on Aug.7, 2014) in U.S. Provisional Application No. 62/046,059, filed Sep. 4,2014. Table 4 provided in U.S. Provisional Application No. 62/046,059 isincorporated by reference herein.

DETAILED DESCRIPTION

The HIV-1 Env trimer undergoes a dramatic structural rearrangementbetween its prefusion mature closed conformation and the CD4-bound openconformation (see Example 1, below). As shown in FIGS. 1-3 , in theprefusion mature closed conformation, the HIV-1 Env trimer includes aV1V2 domain “cap” at its membrane distal apex, with the V1V2 domain ofeach gp120-gp41 protomer in the trimer coming together at the membranedistal apex. At the membrane proximal aspect, the HIV-1 Env ectodomaintrimer includes distinct α6 and α7 helices. CD4 binding causes changesin the conformation of the HIV-1 Env ectodomain trimer, includingdisruption of the V1V1 domain cap, which “opens” as each V1V2 domainmoves outward from the longitudinal axis of the Env trimer following CD4binding, and formation of the HR1 helix, which includes both the α6 andα7 helices (which are no longer distinct, see FIG. 3D). Theseconformational changes bring the N-terminus of the fusion peptide withinclose proximity of the target cell membrane, and expose “CD4-induced”epitopes (such as the 17b epitope) that are present in the CD4-boundopen conformation, but not the mature closed conformation, of the HIV-1Env ectodomain trimer.

Thus, the membrane distal and membrane proximal aspects of the HIV-1 Envectodomain trimer in its prefusion mature closed conformation includeseveral distinct structural elements that are absent from thecorresponding regions of the HIV-1 Env ectodomain trimer in itsCD4-bound open conformation. Amino acid positions (and sequences)corresponding to these regions are indicated in FIGS. 3 and 11 .

Notably, in a previously identified HIV-1 Env ectodomain trimer(BG505.SOSIP, described in more detail in Example 1), CD4 triggeredrecognition by ineffective antibodies so that their average binding wastighter than that of broadly neutralizing antibodies (see Example 2,FIG. 33A). Such CD4 triggering makes HIV-1 Env ectodomain trimers thatcannot resist conformational changes to the CD-bound open conformationless desirable as an immunogen: in primates, such immunogens would bindCD4 in vivo and would thus be expected to elicit production of primarilyineffective antibodies against highly immunogenic CD4-induced epitopes.

Accordingly, recombinant HIV-1 Env proteins are provided that arestabilized or “locked” in the prefusion mature closed conformation.Using structure-guided design, positions of the HIV-1 Env protein weretargeted for modification (e.g., amino acid substitution) to hinder orprevent the HIV-1 Env ectodomain trimer from transitioning from theprefusion mature closed conformation to CD4-bound open conformations.These recombinant HIV-1 Env ectodomain trimers resist transition to theCD4-bound open state of HIV-1 Env, and thus will retain the prefusionmature closed conformation when used as an immunogen to generate animmune response to HIV-1 Env in a subject expressing CD4, such as ahuman.

I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

17b: A monoclonal antibody that specifically binds to a CD4-inducedepitope on the HIV-1 Env ectodomain trimer, that is, CD4 binding causesa conformation change in the HIV-1 Env ectodomain trimer that exposesthe 17b epitope. Thus, 17b mAb is a “CD4-induced” antibody. The 17bantibody does not specifically bind to the HIV-1 Env ectodomain trimerin its prefusion mature closed conformation. The person of ordinaryskill in the art is familiar with monoclonal antibody 17b and withmethods of producing this antibody (see, for example, Kwong et al., J.Biol. Chem., 274, 4115-4123, 1999, which is incorporated by referenceherein). The amino acid sequences of the heavy and light variableregions of the 17b antibody are known and have been deposited in GenBankas Nos. 1G9N_H (17b V_(H)) and 1G9N_L (17b V_(L)), each of which isincorporated by reference herein as present in the database on Jun. 20,2014).

35O22: A neutralizing monoclonal antibody that specifically binds to anepitope on the membrane-proximal region of HIV-1 Env including residuesof both gp120 and gp41. The amino acid sequences of the heavy and lightvariable regions of the 35O22 antibody are set forth as SEQ ID NOs: 2099and 2100, respectively, and can be used to generate an antibody with the35O22 antigen binding domain.

447-52D: A monoclonal antibody that specifically binds to the V3 loop ofHIV-1 Env. The person of ordinary skill in the art is familiar withmonoclonal antibody 447-52D and with methods of producing this antibody(see, for example, Stanfield et al., Structure, 12, 193-204, which isincorporated by reference herein). The amino acid sequences of the heavyand light variable regions of the 447-52D antibody are known and havebeen deposited in the Protein Data Bank as Nos. 1Q1J_H (447-52D V_(H))and 1Q1J_L (447-52D V_(L)), each of which is incorporated by referenceherein as present in the database on Jun. 20, 2014).

Adjuvant: A vehicle used to enhance antigenicity. In some embodiments,an adjuvant can include a suspension of minerals (alum, aluminumhydroxide, or phosphate) on which antigen is adsorbed; or water-in-oilemulsion, for example, in which antigen solution is emulsified inmineral oil (Freund incomplete adjuvant), sometimes with the inclusionof killed mycobacteria (Freund's complete adjuvant) to further enhanceantigenicity (inhibits degradation of antigen and/or causes influx ofmacrophages). Immunostimulatory oligonucleotides (such as thoseincluding a CpG motif) can also be used as adjuvants. Adjuvants includebiological molecules (a “biological adjuvant”), such as costimulatorymolecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-likereceptor (TLR) agonists, such as TLR-9 agonists. In some embodiments,the Adjuplex™ (Advanced BioAdjuvants) can be used with any of therecombinant HIV-1 Env ectodomain trimers to elicit an immune response toHIV-1 Env. The person of ordinary skill in the art is familiar withadjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and DeliverySystems. Wiley-Interscience, 2007). Adjuvants can be used in combinationwith the disclosed immunogens.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition (such as a compositionincluding a disclosed immunogen) is administered by introducing thecomposition into a vein of the subject. Exemplary routes ofadministration include, but are not limited to, oral, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for inhibiting HIV infection in a subject. Agentsinclude proteins, nucleic acid molecules, compounds, small molecules,organic compounds, inorganic compounds, or other molecules of interest.An agent can include a therapeutic agent (such as an anti-retroviralagent), a diagnostic agent or a pharmaceutical agent. In someembodiments, the agent is a protein agent (such as a recombinant HIV-1Env polypeptide or immunogenic fragment thereof), or an anti-viralagent. The skilled artisan will understand that particular agents may beuseful to achieve more than one result.

Amino acid substitutions: The replacement of one amino acid in apolypeptide with a different amino acid or with no amino acid (i.e., adeletion). In some examples, an amino acid in a polypeptide issubstituted with an amino acid from a homologous polypeptide, forexample, and amino acid in a recombinant Clade A HIV-1 Env polypeptidecan be substituted with the corresponding amino acid from a Clade BHIV-1 Env polypeptide.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen)such as HIV-1 gp120, an antigenic fragment thereof, or a dimer ormultimer of the antigen. The term “antibody” is used herein in thebroadest sense and encompasses various antibody structures, includingbut not limited to monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments, so long as they exhibit the desired antigen-binding activity.

Non-limiting examples of antibodies include, for example, intactimmunoglobulins and variants and fragments thereof known in the art thatretain binding affinity for the antigen. Examples of antibody fragmentsinclude but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); and multispecific antibodies formed from antibody fragments.Antibody fragments include antigen binding fragments either produced bythe modification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable domaingenes. There are two types of light chain, lambda (k) and kappa (x).There are five main heavy chain classes (or isotypes) which determinethe functional activity of an antibody molecule: IgM, IgD, IgG, IgA andIgE.

Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space. The CDRs areprimarily responsible for binding to an epitope of an antigen.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which nucleic acid encoding the lightand heavy chains of a single antibody have been transfected, or aprogeny thereof. Monoclonal antibodies are produced by methods known tothose of skill in the art, for instance by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells. These fused cells and their progeny are termed “hybridomas.” Insome examples monoclonal antibodies are isolated from a subject.Monoclonal antibodies can have conservative amino acid substitutionswhich have substantially no effect on antigen binding or otherimmunoglobulin functions. (See, for example, Harlow & Lane, Antibodies,A Laboratory Manual, 2^(nd) ed. Cold Spring Harbor Publications, NewYork (2013).)

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedHIV antigens. Examples of antigens include, but are not limited to,polypeptides, peptides, lipids, polysaccharides, combinations thereof(such as glycopeptides) and nucleic acids containing antigenicdeterminants, such as those recognized by an immune cell. In someexamples, antigens include peptides derived from a pathogen of interest,such as HIV. An antigen can include one or more epitopes.

Anti-retroviral agent: An agent that specifically inhibits a retrovirusfrom replicating or infecting cells. Non-limiting examples ofantiretroviral drugs include entry inhibitors (e.g., enfuvirtide), CCR5receptor antagonists (e.g., aplaviroc, vicriviroc, maraviroc), reversetranscriptase inhibitors (e.g., lamivudine, zidovudine, abacavir,tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g.,lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturationinhibitors (e.g., alpha interferon, bevirimat and vivecon).

Anti-retroviral therapy (ART): A therapeutic treatment for HWV infectioninvolving administration of at least one anti-retroviral agents (e.g.,one, two, three or four anti-retroviral agents) to an HIV infectedindividual during a course of treatment. Non-limiting examples ofantiretroviral agents include entry inhibitors (e.g., enfuvirtide), CCR5receptor antagonists (e.g., aplaviroc, vicriviroc, maraviroc), reversetranscriptase inhibitors (e.g., lamivudine, zidovudine, abacavir,tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g.,lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturationinhibitors (e.g., alpha interferon, bevirimat and vivecon). One exampleof an ART regimen includes treatment with a combination of tenofovir,emtricitabine and efavirenz. In some examples, ART include Highly ActiveAnti-Retroviral Therapy (HAART).

Atomic coordinates or structure coordinates: Mathematical coordinatesderived from mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) such as an antigen, or an antigen in complex with an antibody.In some examples that antigen can be HWV-1 Env polypeptide (for examplestabilized in a prefusion conformation by binding to aprefusion-specific antibody, or by introduction of stabilizingmodifications) in a crystal. The diffraction data are used to calculatean electron density map of the repeating unit of the crystal. Theelectron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal. In one example,the term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays, such as by the atoms of aHIV-1 Env polypeptide in crystal form.

Those of ordinary skill in the art understand that a set of structurecoordinates determined by X-ray crystallography is not without standarderror. For the purpose of this disclosure, any set of structurecoordinates that have a root mean square deviation of protein backboneatoms (N, Cα, C and O) of less than about 1.0 Angstroms whensuperimposed, such as about 0.75, or about 0.5, or about 0.25 Angstroms,using backbone atoms, shall (in the absence of an explicit statement tothe contrary) be considered identical.

Cavity-filling amino acid substitution: An amino acid substitution thatfills a cavity within the protein core of the HIV-1 Env ectodomaintrimer. Cavities are essentially voids within a folded protein whereamino acids or amino acid side chains are not present. In severalembodiments, a cavity filling amino acid substitution is introduced tofill a cavity in the HIV-1 Env ectodomain trimer core present in theprefusion mature closed conformation of HIV-1 Env ectodomain trimer thatcollapses (e.g., has reduced volume) upon transition to the CD4-boundopen conformation.

CD4: Cluster of differentiation factor 4 polypeptide; a T-cell surfaceprotein that mediates interaction with the MHC class II molecule. CD4also serves as the primary receptor site for HIV on T-cells during HIVinfection. CD4 is known to bind to gp120 from HIV-1 Env. The sequence ofthe CD4 precursor has a hydrophobic signal peptide, an extracellularregion of approximately 370 amino acids, a highly hydrophobic stretchwith significant identity to the membrane-spanning domain of the classII MHC beta chain, and a highly charged intracellular sequence of 40resides (Maddon, Cell 42:93, 1985). The amino acid sequence of human CD4is deposited in GenBank as No. P01730.1. Several embodiments utilizesoluble CD4 (sCD4), which includes the extracellular domain of CD4(without the signal peptide), approximately CD4 amino acids 26-390(e.g., SEQ ID NO: 2118). Soluble CD4 can be obtained commercially (e.g.,from Mybiosource); methods of its production are well known in the art.

CD4-induced antibody: An antibody that binds to an epitope present onthe CD4-bound open conformation of the HIV-1 Env ectodomain trimer, butnot present on the mature closed conformation of the HIV-1 Envectodomain trimer. An example of a CD4-induced antibody is 17b mAb.

Conditions sufficient to form an immune complex: Conditions which allowan antibody or antigen binding fragment thereof to bind to its cognateepitope to a detectably greater degree than, and/or to the substantialexclusion of, binding to substantially all other epitopes. Conditionssufficient to form an immune complex are dependent upon the format ofthe binding reaction and typically are those utilized in immunoassayprotocols or those conditions encountered in vivo. See Harlow & Lane,Antibodies, A Laboratory Manual, 2^(nd) ed. Cold Spring HarborPublications, New York (2013) for a description of immunoassay formatsand conditions. The conditions employed in the methods are“physiological conditions” which include reference to conditions (e.g.,temperature, osmolarity, pH) that are typical inside a living mammal ora mammalian cell. While it is recognized that some organs are subject toextreme conditions, the intra-organismal and intracellular environmentnormally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typicallypH 6.5 to 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. Osmolarity is within therange that is supportive of cell viability and proliferation.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease a function ofa protein, such as the ability of the protein to induce an immuneresponse when administered to a subject. The term conservative variationalso includes the use of a substituted amino acid in place of anunsubstituted parent amino acid. Furthermore, one of ordinary skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (for instance less than 5%, in some embodiments less than 1%) inan encoded sequence are conservative variations where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of the recombinant Env protein, such as the ability to inducean immune response when administered to a subject. For instance, if anamino acid residue is essential for a function of the protein, even anotherwise conservative substitution may disrupt that activity. Thus, aconservative substitution does not alter the basic function of a proteinof interest.

Circular permutant: A modified recombinant protein in which theconnections between different regions of a protein tertiary structure ismodified, so that the relative order of different regions in the primarysequence is altered, but the placement of the regions in the tertiarystructure is preserved. For example, with a 4-stranded antiparallelsheet, with strand A, B, C and D, which has the following N and Ctermini and connectivity:

Nterm-strand A-linker-strand B-linker-strand C-linker-strand D-Cterm,circular permutants of the 4 strands, A, B, C and D by altering linkerconnection between strands can include:

-   -   Permutation with N- and C-termini altered:

Nterm-strand C-linker-strand D-linker-strand A-linker-strand B-Cterm

-   -   Permutation with N terminus preserved:

Nterm-strand A-linker-strand D-linker-strand C-linker-strand B-C term

-   -   Permutation with C terminus preserved:

Nterm-strand C-linker-strand B-linker-strand A-linker-strand D-C term.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as apeptide, that contacts another polypeptide. Contacting can also includecontacting a cell for example by placing a polypeptide in directphysical association with a cell.

Control: A reference standard. In some embodiments, the control is anegative control sample obtained from a healthy patient. In otherembodiments, the control is a positive control sample obtained from apatient diagnosed with HIV infection. In still other embodiments, thecontrol is a historical control or standard reference value or range ofvalues (such as a previously tested control sample, such as a group ofHIV patients with known prognosis or outcome, or group of samples thatrepresent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a polypeptide(such as a recombinant HIV-1 Env ectodomain or immunogenic fragmentthereof) that includes a sequence that is degenerate as a result of thegenetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences encoding a peptide are included as long as the amino acidsequence of the peptide encoded by the nucleotide sequence is unchanged.

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan and may besupplemented with the protocols and reagents disclosed herein. Forexample, included herein are methods of detecting the level of a proteinin a sample or a subject.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. An antibody can bind toa particular antigenic epitope, such as an epitope on HIV-1 Env.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its mRNA is translated into an amino acidsequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Expression control sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signal forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as μL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specificnucleic acid sequences that allow phenotypic selection of thetransformed cells.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Heterologous: Originating from a different genetic source. A nucleicacid molecule that is heterologous to a cell originated from a geneticsource other than the cell in which it is expressed. In one specific,non-limiting example, a heterologous nucleic acid molecule encoding arecombinant HIV-1 Env polypeptide is expressed in a cell, such as amammalian cell. Methods for introducing a heterologous nucleic acidmolecule in a cell or organism are well known in the art, for exampletransformation with a nucleic acid, including electroporation,lipofection, particle gun acceleration, and homologous recombination.

F105: A monoclonal antibody that specifically binds to a conformationalepitope on HIV-1 Env that is not present on the prefusion mature closedconformation. The F105 antibody does not specifically bind to HIV-1 Envin its prefusion mature closed conformation. The person of ordinaryskill in the art is familiar with monoclonal antibody F105 and withmethods of producing this antibody (see, for example, Posner et al. JAcquired Immune Defic Syndr 6:7-14, 1993; which is incorporated byreference herein). The amino acid sequences of the heavy and lightvariable regions of the F105 antibody are known and have been depositedin the Protein Data Bank (PDB) as No. 1U6A_H (F105 V_(H)) and 1U6A-L(F105 V_(L)), each of which is incorporated by reference herein aspresent in the database on Jun. 20, 2014).

Ferritin: A protein that stores iron and releases it in a controlledfashion. The protein is produced by almost all living organisms.Ferritin polypeptides assemble into a globular protein complex of 24protein subunits, each of the 24 subunits includes a single ferritinpolypeptide. In some examples, ferritin is used to form a nanoparticlepresenting antigens on its surface, for example, an HIV antigen.

Foldon domain: An amino acid sequence that naturally forms a trimericstructure. In some examples, a Foldon domain can be included in theamino acid sequence of a disclosed recombinant protein so that theantigen will form a trimer. In one example, a Foldon domain is the T4Foldon domain including the amino acid sequence set forth as(GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 578). Several embodimentsinclude a Foldon domain that can be cleaved from a purified protein, forexample by incorporation of a thrombin cleave site adjacent to theFoldon domain that can be used for cleavage purposes.

Glycosylation site: An amino acid sequence on the surface of apolypeptide, such as a protein, which accommodates the attachment of aglycan. An N-linked glycosylation site is triplet sequence of NX(S/T) inwhich N is asparagine, X is any residues except proline, and (S/T) is aserine or threonine residue. A glycan is a polysaccharide oroligosaccharide. Glycan may also be used to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid, or aproteoglycan.

Homologous proteins: Proteins that have a similar structure andfunction, for example, proteins from two or more species or viralstrains that have similar structure and function in the two or morespecies or viral strains. For example a HIV-1 Env protein from a Clade Avirus is a homologous protein to a HIV-1 Env protein from Clade B virus.Homologous proteins share similar protein folding characteristics andcan be considered structural homologs.

Homologous proteins typically share a high degree of sequenceconservation, such as at least 80%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence conservation, and a highdegree of sequence identity, such as at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, or at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequenceidentity.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Human Immunodeficiency Virus (HIV): A retrovirus that causesimmunosuppression in humans (HIV disease), and leads to a diseasecomplex known as the acquired immunodeficiency syndrome (AIDS). “HIVdisease” refers to a well-recognized constellation of signs and symptoms(including the development of opportunistic infections) in persons whoare infected by an HIV virus, as determined by antibody or western blotstudies. Laboratory findings associated with this disease include aprogressive decline in T cells. HIV includes HIV type 1 (HIV-1) and HIVtype 2 (HIV-2). Related viruses that are used as animal models includesimian immunodeficiency virus (SIV), and feline immunodeficiency virus(FIV). Treatment of HIV-1 with HAART has been effective in reducing theviral burden and ameliorating the effects of HIV-1 infection in infectedindividuals.

HIV-1 broadly neutralizing antibody: An antibody that reduces theinfectious titer of HIV-1 by binding to and inhibiting the function ofrelated HIV-1 antigens, such as antigens that share at least 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity with antigenic surface ofthe antigen. In some embodiments, broadly neutralizing antibodies to HIVare distinct from other antibodies to HIV in that they neutralize a highpercentage (such as at least 50% or at least 80%) of the many types ofHIV in circulation. Non-limiting examples of HIV-1 broadly neutralizingantibodies include PGT122, VRC01, and 35O22.

HIV envelope protein (Env): The HIV envelope protein is initiallysynthesized as a precursor protein of 845-870 amino acids in size,designated gp160. Individual gp160 polypeptides form a homotrimer andundergo glycosylation within the Golgi apparatus as well as processingto remove the signal peptide, and cleavage by a cellular proteasebetween approximately positions 511/512 to generate separate gp120 andgp41 polypeptide chains, which remain associated as gp120-gp41 protomerswithin the homotrimer. The ectodomain (that is, the extracellularportion) of the HIV-1 Env trimer undergoes several structuralrearrangements from a prefusion mature (cleaved) closed conformationthat evades antibody recognition, through intermediate conformationsthat bind to receptors CD4 and co-receptor (either CCR5 or CXCR4), to apostfusion conformation. The HIV-1 Env ectodomain includes the gp120protein (approximately HIV-1 Env positions 31-511) and the gp41ectodomain (approximately HIV-1 Env positions 512-644). An HIV-1 Envectodomain trimer includes a protein complex of three HIV-1 Envectodomains. Mature gp120 includes approximated HIV-1 Env residues31-511, contains most of the external, surface-exposed, domains of theHIV-1 Env trimer, and it is gp120 which binds both to cellular CD4receptors and to cellular chemokine receptors (such as CCR5). A maturegp120 polypeptide is an extracellular polypeptide that interacts withthe gp41 ectodomain to form an HIV-1 Env protomer that trimerizes toform the HIV-1 Env trimer. The mature gp120 wild-type polypeptide isheavily N-glycosylated, giving rise to an apparent molecular weight of120 kD. Native gp120 includes five conserved regions (C1-C5) and fiveregions of high variability (V1-V5). See FIG. 11 for an illustration ofgp120 primary and secondary structures.

Variable region 1 and Variable Region 2 (V1/V2 domain) of gp120 arecomprised of ˜50-90 residues which contain two of the most variableportions of HIV-1 (the V1 domain and the V2 loop), and one in tenresidues of the V1/V2 domain are N-glycosylated. Despite the diversityand glycosylation of the V1/V2 domain, a number of broadly neutralizinghuman antibodies have been identified that target this region, includingPG9 and PGT122. In some examples the V1/V2 domain includes gp120positions 126-196. Variable region 3 (V3) of gp120 includesapproximately 35-45 amino acids. In some examples the V1/V2 domainincludes gp120 positions 296-331. Mature gp41 includes approximatelyHIV-1 Env residues 512-860, and includes cytosolic-, transmembrane- andecto-domains. The gp41 ectodomain (including approximately HIV-1 Envresidues 512-644) can interact with gp120 to form an HIV-1 Env protomerthat trimerizes to form the HIV-1 Env trimer. See FIG. 3 for anillustration of the primary and secondary structure of a gp41ectodomain.

The numbering used in the disclosed HIV-1 Env proteins and fragmentsthereof is relative to the HXB2 numbering scheme as set forth inNumbering Positions in HIV Relative to HXB2CG Bette Korber et al., HumanRetroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acidand Amino Acid Sequences. Korber et al., Eds. Theoretical Biology andBiophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex.,which is incorporated by reference herein in its entirety.

In one example, an HIV-1 Env protein is from the BG505 strain of HIV,which is a Clade A HIV-1 virus isolated from a six-week old HIV-1infected infant. The amino acid sequence of BG505 Env protein is known(see, e.g., GenBank accession no. ABA61516, incorporated by referenceherein as present in the database on Jun. 20, 2014), and set forth asSEQ ID NO: 2.

HIV-1 Env ectodomain trimer prefusion mature closed conformation: Astructural conformation adopted by the ectodomain of the HIV-1 Envectodomain trimer after cellular processing to a mature prefusion statewith distinct gp120 and gp41 polypeptide chains, and before specificbinding to the CD4 receptor. In the prefusion mature closedconformation, the HIV-1 Env ectodomain trimer includes a V1V2 domain“cap” at its membrane distal apex, with the V1V2 domain of each Envprotomer in the trimer coming together at the membrane distal apex. Atthe membrane proximal aspect, the HIV-1 Env ectodomain trimer includesdistinct α6 and α7 helices. CD4 binding to the Env trimer causes changesin the conformation of the HIV-1 Env ectodomain trimer, includingdisruption of the V1V1 domain cap, which “opens” as each V1V2 domainmoves outward from the longitudinal axis of the Env trimer, andformation of the HR1 helix, which includes both the α6 and α7 helices(which are no longer distinct, see FIG. 3D). These conformationalchanges bring the N-terminus of the fusion peptide within closeproximity of the target cell membrane, and expose “CD4-induced” epitopes(such as the 17b epitope) that are present in the CD4-bound openconformation, but not the mature closed conformation, of the HIV-1 Envectodomain trimer. The three-dimensional structure of an exemplary HIV-1Env ectodomain trimer in the prefusion mature closed conformation isdisclosed herein (see Example 1).

HIV-1 Env ectodomain trimer stabilized in a prefusion mature closedconformation: A HIV-1 Env ectodomain trimer having a prefusion matureclosed conformation and including one or more amino acid substitutions,deletions, or insertions compared to a native HIV-1 Env sequence thatprovide for increased retention of the prefusion mature closedconformation upon CD4 binding compared to a corresponding native HIV-1Env sequence. In some embodiments, the HIV-1 Env ectodomain trimer caninclude one or more cysteine substitutions that allow formation of anon-natural disulfide bond that stabilizes the HIV-1 Env ectodomaintrimer in its prefusion mature closed conformation.

A HIV-1 Env ectodomain trimer stabilized in the prefusion mature closedconformation has at least 90% (such as at least 95% or at least 99%)reduced transition to the CD4-bound open conformation upon CD4 bindingcompared to a corresponding native HIV-1 Env sequence. The“stabilization” of the prefusion mature closed conformation by the oneor more amino acid substitutions, deletions, or insertions can be, forexample, energetic stabilization (for example, reducing the energy ofthe prefusion mature closed conformation relative to the CD4-bound openconformation) and/or kinetic stabilization (for example, reducing therate of transition from the prefusion mature closed conformation to theprefusion mature closed conformation). Additionally, stabilization ofthe HIV-1 Env ectodomain trimer in the prefusion mature closedconformation can include an increase in resistance to denaturationcompared to a corresponding native HIV-1 Env sequence.

Methods of determining if a HIV-1 Env ectodomain trimer is in theprefusion mature closed conformation are provided herein, and include(but are not limited to) negative stain electron microscopy and antibodybinding assays using a prefusion mature closed conformation specificantibody, such as VRC26 or PGT145. Methods of determining if a HIV-1 Envectodomain trimer is in the CD4-bound open conformation are alsoprovided herein, and include (but are not limited to) negative stainelectron microscopy and antibody binding assays using a CD4-bound openconformation specific antibody, such as 17b, which binds to aCD4-induced epitope. Transition from the prefusion mature closedconformation upon CD4 binding can be assayed, for example, by incubatinga HIV-1 Env ectodomain trimer of interest that is in the prefusionmature closed conformation with a molar excess of CD4, and determiningif the HIV-1 Env ectodomain trimer retains the prefusion mature closedconformation (or transitions to the CD4-bound open conformation) bynegative stain electron microscopy analysis, or antigenic analysis.

HIV-1 gp140: A recombinant HIV Env polypeptide including gp120 and thegp41 ectodomain, but not the gp41 transmembrane or cytosolic domains.HIV-1 gp140 polypeptides can trimerize to form a soluble HIV-1 Envectodomain trimer.

HIV-1 gp145: A recombinant HIV Env polypeptide including gp120, the gp41ectodomain, and the gp41 transmembrane domain. HIV-1 gp145 polypeptidescan trimerize to form membrane bound HIV-1 Env ectodomain trimers.

HXB2 numbering system: A reference numbering system for HIV protein andnucleic acid sequences, using the HIV-1 HXB2 strain sequences as areference for all other HIV-1 strain sequences. The person of ordinaryskill in the art is familiar with the HXB2 numbering system, and thissystem is set forth in “Numbering Positions in HIV Relative to HXB2CG,”Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation andAnalysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L,Foley B, Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds.Theoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, N. Mex., which is incorporated by referenceherein in its entirety. Unless context indicates otherwise, thenumbering used in HIV-1 polypeptides disclosed herein is relative to theHXB2 numbering scheme. For reference, the amino acid sequence of HIV-1Env of HXB2 is set forth as SEQ ID NO: 1 (GENBANK® Accession No. K03455,incorporated by reference herein as present in the database on Jun. 20,2014).

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies. “Priming an immune response” refers to pre-treatment of asubject with an adjuvant to increase the desired immune response to alater administered immunogenic agent. “Enhancing an immune response”refers to co-administration of an adjuvant and an immunogenic agent,wherein the adjuvant increases the desired immune response to theimmunogenic agent compared to administration of the immunogenic agent tothe subject in the absence of the adjuvant.

Immunogen: A protein or a portion thereof that is capable of inducing animmune response in a mammal, such as a mammal infected or at risk ofinfection with a pathogen. Administration of an immunogen can lead toprotective immunity and/or proactive immunity against a pathogen ofinterest. In some examples, an immunogen comprises a recombinant HIV-1Env ectodomain trimer as disclosed herein.

Immunogenic composition: A composition comprising an immunogenicpolypeptide, or a nucleic acid molecule or vector encoding animmunogenic polypeptide that induces a measurable CTL response againstthe immunogenic polypeptide, or induces a measurable B cell response(such as production of antibodies) against the immunogenic polypeptide.In one example, an “immunogenic composition” is a composition thatincludes a disclosed recombinant HIV-1 Env ectodomain trimer orimmunogenic fragment thereof, that induces a measurable CTL responseagainst an HIV-1 virus, or induces a measurable B cell response (such asproduction of antibodies) against a HIV-1. It further refers to isolatednucleic acids encoding an antigen, such as a nucleic acid that can beused to express the antigen (and thus be used to elicit an immuneresponse against this peptide).

For in vitro use, an immunogenic composition may comprise or consist ofthe isolated protein or nucleic acid molecule encoding the protein. Forin vivo use, the immunogenic composition will typically include theprotein or nucleic acid molecule in a pharmaceutically acceptablecarrier and may also include other agents, such as an adjuvant. Anyparticular protein, such as a disclosed recombinant HIV-1 Env ectodomaintrimer or a nucleic acid encoding the protein, can be readily tested forits ability to induce a CTL or B cell response by art-recognized assays.Immunogenic compositions can include adjuvants, which are well known toone of skill in the art.

Immunogenic polypeptide: A polypeptide which comprises anallele-specific motif, an epitope or other sequence such that thepolypeptide will bind an MHC molecule and induce an immune response,such as a cytotoxic T lymphocyte (“CTL”) response, and/or a B cellresponse (for example, antibody production), and/or a T-helperlymphocyte response against the antigen from which the immunogenicpolypeptide is derived.

Isolated: An “isolated” biological component (such as a protein, forexample a disclosed immunogen or nucleic acid encoding such an antigen)has been substantially separated or purified away from other biologicalcomponents, such as other biological components in which the componentnaturally occurs, such as other chromosomal and extrachromosomal DNA,RNA, and proteins. Proteins, peptides and nucleic acids that have been“isolated” include proteins purified by standard purification methods.The term also embraces proteins or peptides prepared by recombinantexpression in a host cell as well as chemically synthesized proteins,peptides and nucleic acid molecules. Isolated does not require absolutepurity, and can include protein, peptide, or nucleic acid molecules thatare at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%,99%, or even 99.9% isolated. The HIV-1 Env proteins herein that arestabilized in a prefusion mature closed conformation can be isolatedfrom HIV-1 Env proteins in a prefusion CD4 bound conformation, forexample, can be at least 80% isolated, at least 90%, 95%, 98%, 99%, oreven 99.9% isolated from HIV-1 Env proteins in a prefusion CD4 boundconformation.

K_(D): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody or antigenbinding fragment and an immunogen (such as HIV-1 Env polypeptide) it isthe concentration of the individual components of the bimolecularinteraction divided by the concentration of the complex.

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule, for example, to link a carrier molecule toa immunogenic polypeptide. Non-limiting examples of peptide linkersinclude glycine-serine linkers, such as a (GGGGS), linker or a 10 aminoacid glycine-serine linker. Unless context indicates otherwise,reference to “linking” a first polypeptide and a second polypeptide (orto two polypeptides “linked” together) refers to covalent linkage bypeptide bond, or (if a peptide linker is involved) covalent linkage ofthe first and second polypeptides to the N and C termini of a peptidelinker. Thus, reference to a gp120 polypeptide “linked” to a gp41ectodomain by a peptide linker indicates that the gp120 polypeptide andthe gp41 ectodomain are linked to opposite ends of the peptide linker bypeptide bonds. Typically, such linkage is accomplished using molecularbiology techniques to genetically manipulate DNA encoding the firstpolypeptide linked to the second polypeptide by the peptide linker.

The terms “conjugating,” “joining,” “bonding,” can refer to making twomolecules into one contiguous molecule; for example, joining twopolypeptides into one contiguous polypeptide, or covalently attaching acarrier molecule or other molecule to an immunogenic polypeptide, suchas an recombinant HIV-1 Env ectodomain as disclosed herein. Theconjugate can be either by chemical or recombinant means. “Chemicalmeans” refers to a reaction, for example, between the immunogenicpolypeptide moiety and the carrier molecule such that there is acovalent bond formed between the two molecules to form one molecule.

Neutralizing antibody: An antibody which reduces the infectious titer ofan infectious agent by binding to a specific antigen on the infectiousagent. In some examples the infectious agent is a virus. In someexamples, an antibody that is specific for HIV-1 Env neutralizes theinfectious titer of HIV. A “broadly neutralizing antibody” is anantibody that binds to and inhibits the function of related antigens,such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99%identity antigenic surface of antigen. With regard to an antigen from apathogen, such as a virus, the antibody can bind to and inhibit thefunction of an antigen from more than one class and/or subclass of thepathogen. For example, with regard to a human immunodeficiency virus,the antibody can bind to and inhibit the function of an antigen, such asHIV-1 Env from more than one clade. In one embodiment, broadlyneutralizing antibodies to HIV are distinct from other antibodies to HIVin that they neutralize a high percentage of the many types of HIV incirculation.

Native protein, sequence, or di-sulfide bond: An polypeptide, sequenceor di-sulfide bond that has not been modified, for example by selectivemutation. For example, selective mutation to focus the antigenicity ofthe antigen to a target epitope, or to introduce a di-sulfide bond intoa protein that does not occur in the native protein. Native protein ornative sequence are also referred to as wild-type protein or wild-typesequence. A non-native di-sulfide bond is a disulfide bond that is notpresent in a native protein, for example a di-sulfide bond that forms ina protein due to introduction of one or more cysteine residues into theprotein by genetic engineering.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. It will beunderstood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“Nucleotide” includes, but is not limited to, a monomer that includes abase linked to a sugar, such as a pyrimidine, purine or syntheticanalogs thereof, or a base linked to an amino acid, as in a peptidenucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. Anucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

PGT121, PGT122, and PGT123: A family of neutralizing monoclonalantibodies that specifically bind to the V1/V2 and V3 regions of HIV-1Env and can inhibit HIV-1 infection of target cells. The person ofordinary skill in the art is familiar with the PGT121, PGT122, andPGT123 mAbs and with methods of producing them (see, for example, Walkeret al., Nature, 477:466-470, 2011, and Int. Pub. No. WO 2012/030904,each of which is incorporated by reference herein). The amino acidsequences of the heavy and light variable regions of the PGT121, PGT122,and PGT123 antibodies are known and have been deposited in GenBank asNos. AEN14390.1 (PGT121 V_(H)), AEN14407.1 (PGT121 V_(L)), JN201895.1(PGT122 V_(H)), JN201912.1 (PGT122 V_(L)), JN201896.1 (PGT123 V_(H)),and JN201913.1 (PGT123 V_(L)), each of which is incorporated byreference herein as present in the database on Jun. 20, 2014)

PGT141, PGT142, PGT143, and PGT145: A family of broadly neutralizingmonoclonal antibodies that specifically bind to the V1/V2 domain of theHIV-1 Env ectodomain trimer in its prefusion mature closed conformation,and which can inhibit HIV-1 infection of target cells. The person ofordinary skill in the art is familiar with the PGT141, PGT142, PGT143,and PGT145 mAbs and with methods of producing them (see, for example,Walker et al., Nature, 477:466-470, 2011, and Int. Pub. No.WO2012/030904, each of which is incorporated by reference herein). Theamino acid sequences of the heavy and light variable regions of thePGT141, PGT142, PGT143, PGT144, and PGT145 mAbs are known and have beendeposited in GenBank as Nos. JN201906.1 (PGT141 V_(H)), JN201923.1(PGT141 V_(L)), JN201907.1 (PGT142 V_(H)), JN201924.1 (PGT142 V_(L)),JN201908.1 (PGT143 V_(H)), JN201925.1 (PGT143 V_(L)), JN201909.1 (PGT144V_(H)), JN201926.1 (PGT144 V_(L)), JN201910.1 (PGT145 V_(H)), andJN201927.1 (PGT145 V_(L)), each of which is incorporated by referenceherein as present in the database on Jun. 20, 2014).

PGT151: A broadly neutralizing monoclonal antibody that specificallybind to the gp120/gp41 interface of HIV-1 Env in its prefusion mature(cleaved) conformation, and which can inhibit HIV-1 infection of targetcells. The person of ordinary skill in the art is familiar with thePGT151 antibody and with methods of producing this antibody (see, forexample, Blattner et al., Immunity, 40, 669-680, 2014, and Falkowska etal., Immunity, 40, 657-668, 2014, each of which is incorporated byreference herein). The amino acid sequences of the heavy and lightvariable regions of the PGT151 mAb are known and have been deposited inGenBank as Nos. KJ700282.1 (PGT151 V_(H)) and KJ700290.1 (PGT151 V_(L)),each of which is incorporated by reference herein as present in thedatabase on Jun. 20, 2014).

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to induce the desiredanti-HIV-1 immune response. It may also be accompanied by medicationsfor its use for treatment purposes. The unit dosage form may be, forexample, in a sealed vial that contains sterile contents or a syringefor injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues. A protein can includemultiple polypeptide chains; for example, mature HIV-1 Env includesgp120 and gp41 polypeptide chains. Additionally, a single contiguouspolypeptide chain of amino acid residues can include multiplepolypeptides. For example, a single chain HIV-1 Env can include a gp120polypeptide linked to a gp41 polypeptide by a peptide linker.

In many instances, one or more polypeptides can fold into a specificthree-dimensional structure including surface-exposed amino acidresidues and non-surface-exposed amino acid residues. In some instancesa protein can include multiple polypeptides that fold together into afunctional unit. For example, the HIV-1 Env protein is composed of threegp120-gp41 protomers that trimerize in to a multimeric protein.“Surface-exposed amino acid residues” are those amino acids that havesome degree of exposure on the surface of the protein, for example suchthat they can contact the solvent when the protein is in solution. Incontrast, non-surface-exposed amino acids are those amino acid residuesthat are not exposed on the surface of the protein, such that they donot contact solution when the protein is in solution. In some examples,the non-surface-exposed amino acid residues are part of the proteincore.

A “protein core” is the interior of a folded protein, which issubstantially free of solvent exposure, such as solvent in the form ofwater molecules in solution. Typically, the protein core ispredominately composed of hydrophobic or apolar amino acids. In someexamples, a protein core may contain charged amino acids, for exampleaspartic acid, glutamic acid, arginine, and/or lysine. The inclusion ofuncompensated charged amino acids (a compensated charged amino can be inthe form of a salt bridge) in the protein core can lead to adestabilized protein. That is, a protein with a lower T_(m) then asimilar protein without an uncompensated charged amino acid in theprotein core. In other examples, a protein core may have a cavity withinthe protein core. Cavities are essentially voids within a folded proteinwhere amino acids or amino acid side chains are not present. Suchcavities can also destabilize a protein relative to a similar proteinwithout a cavity. Thus, when creating a stabilized form of a protein, itmay be advantageous to substitute amino acid residues within the core inorder to fill cavities present in the wild-type protein.

Polypeptide modifications: Polypeptides and peptides, such as therecombinant HIV-1 Env proteins disclosed herein can be modified by avariety of chemical techniques to produce derivatives having essentiallythe same activity as the unmodified peptides, and optionally havingother desirable properties. For example, carboxylic acid groups of theprotein, whether carboxyl-terminal or side chain, may be provided in theform of a salt of a pharmaceutically-acceptable cation or esterified toform a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ whereinR₁ and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to forma heterocyclic ring, such as a 5- or 6-membered ring. Amino groups ofthe peptide, whether amino-terminal or side chain, may be in the form ofa pharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides of this disclosure toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a C- or N-terminal cysteinecan be added to the peptide, so that when oxidized the peptide willcontain a disulfide bond, generating a cyclic peptide. Other peptidecyclizing methods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Prime-boost vaccination: An immunotherapy including administration of afirst immunogenic composition (the primer vaccine) followed byadministration of a second immunogenic composition (the booster vaccine)to a subject to induce an immune response. The primer vaccine and/or thebooster vaccine include a vector (such as a viral vector, RNA, or DNAvector) expressing the antigen to which the immune response is directed.The booster vaccine is administered to the subject after the primervaccine; the skilled artisan will understand a suitable time intervalbetween administration of the primer vaccine and the booster vaccine,and examples of such timeframes are disclosed herein. In someembodiments, the primer vaccine, the booster vaccine, or both primervaccine and the booster vaccine additionally include an adjuvant. In onenon-limiting example, the primer vaccine is a DNA-based vaccine (orother vaccine based on gene delivery), and the booster vaccine is aprotein subunit or protein nanoparticle based vaccine.

Protein nanoparticle: A multi-subunit, protein-based polyhedron shapedstructure. The subunits are each composed of proteins or polypeptides(for example a glycosylated polypeptide), and, optionally of single ormultiple features of the following: nucleic acids, prosthetic groups,organic and inorganic compounds. Non-limiting examples of proteinnanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int.J. Mol. Sci., 12:5406-5421, 2011, incorporated by reference herein),encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. andMol. Biol., 15:939-947, 2008, incorporated by reference herein), SulfurOxygenase Reductase (SOR) nanoparticles (see, e.g., Urich et al.,Science, 311:996-1000, 2006, incorporated by reference herein), lumazinesynthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001) or pyruvate dehydrogenase nanoparticles (see, e.g.,Izard et al., PNAS 96: 1240-1245, 1999, incorporated by referenceherein). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvatedehydrogenase are monomeric proteins that self-assemble into a globularprotein complexes that in some cases consists of 24, 60, 24, 60, and 60protein subunits, respectively. In some examples, ferritin, encapsulin,SOR, lumazine synthase, or pyruvate dehydrogenase monomers are linked toa recombinant HIV-1 Env ectodomain and self-assemble into a proteinnanoparticle presenting the recombinant HIV-1 Env ectodomain on itssurface, which can be administered to a subject to stimulate an immuneresponse to the antigen.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished, for example, theartificial manipulation of isolated segments of nucleic acids, forexample using genetic engineering techniques. A recombinant protein isone that has a sequence that is not naturally occurring or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. In several embodiments, a recombinantprotein is encoded by a heterologous (for example, recombinant) nucleicacid that has been introduced into a host cell, such as a bacterial oreukaryotic cell. The nucleic acid can be introduced, for example, on anexpression vector having signals capable of expressing the proteinencoded by the introduced nucleic acid or the nucleic acid can beintegrated into the host cell chromosome.

Root mean square deviation (RMSD): The square root of the arithmeticmean of the squares of the deviations from the mean. In severalembodiments, RMSD is used as a way of expressing deviation or variationfrom the structural coordinates of a reference three dimensionalstructure. This number is typically calculated after optimalsuperposition of two structures, as the square root of the mean squaredistances between equivalent C. atoms.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA), protein, or combinations thereof, obtainedfrom a subject. Examples include, but are not limited to, peripheralblood, tissue, cells, urine, saliva, tissue biopsy, fine needleaspirate, surgical specimen, and autopsy material.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs, orthologs, or variants of a polypeptide will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a peptide sequence that has 1166matches when aligned with a test sequence having 1554 amino acids is75.0 percent identical to the test sequence (1166 1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

Homologs and variants of a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013). One example of a useful algorithm isPILEUP. PILEUP uses a simplification of the progressive alignment methodof Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used issimilar to the method described by Higgins & Sharp, CABIOS 5:151-153,1989. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

As used herein, reference to “at least 80% identity” (or similarlanguage) refers to “at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or even 100% identity” toa specified reference sequence. As used herein, reference to “at least90% identity” (or similar language) refers to “at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or even 100% identity” toa specified reference sequence.

Single chain HIV-1 Env ectodomain: A recombinant polypeptide includinggp120 and the gp41 ectodomain in a single polypeptide chain. Singlechain HWV-1 Env ectodomains can trimerize to form a trimeric HIV-1 Envectodomain. A single chain HIV-1 Env ectodomain does not include thefurin cleavage site separating gp120 and gp41; therefore, when producedin cells, the Env ectodomain is not cleaved into separate gp120 and gp41ectodomain polypeptides. For example, the gp120 and gp41 proteins can belinked by a peptide linker, or directly linked. In some embodiments, asingle chain HIV-1 Env ectodomain includes a circular permuted HIV-1 Envectodomain. Single chain HIV-1 Env ectodomains are of particularinterest for DNA or vector encoded immunogens, as it can be beneficialin these immunogen format to not need to rely on the presence of furin(or furin-like) protease in a host cell for maturation of separategp120/gp41 polypeptides.

Signal Peptide: A short amino acid sequence (e.g., approximately 18-25amino acids in length) that directs newly synthesized secretory ormembrane proteins to and through membranes (for example, the endoplasmicreticulum membrane). Signal peptides are typically located at theN-terminus of a polypeptide and are removed by signal peptidases afterthe polypeptide has crossed the membrane. Signal peptide sequencestypically contain three common structural features: an N-terminal polarbasic region (n-region), a hydrophobic core, and a hydrophilicc-region). Exemplary signal peptide sequences are set forth as residues1-30 of SEQ ID NO: 1 (HXB2 Env signal peptide) and SEQ ID NO: 2 (BG505Env signal peptide).

Specifically bind: When referring to the formation of anantibody:antigen protein complex, or a protein:protein complex, refersto a binding reaction which determines the presence of a target protein,peptide, or polysaccharide (for example a glycoprotein), in the presenceof a heterogeneous population of proteins and other biologics. Thus,under designated conditions, an particular antibody or protein bindspreferentially to a particular target protein, peptide or polysaccharide(such as an antigen present on the surface of a pathogen, for examplegp120) and does not bind in a significant amount to other proteins orpolysaccharides present in the sample or subject. Specific binding canbe determined by methods known in the art. A first protein or antibodyspecifically binds to a target protein when the interaction has a K_(D)of less than 10⁻⁶ Molar, such as less than 10⁻⁷ Molar, less than 10⁻⁸Molar, less than 10⁻⁹, or even less than 10⁻¹⁰ Molar. In someembodiments, an antibody does not specifically bind to a disclosedrecombinant HIV-1 Env ectodomain trimer if the binding interaction ofthe antibody to trimer has a K_(D) of more than 10⁻⁶ when assayed atstoichiometry of at least one antibody Fab per protomer in the trimer.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In a particular example, the subject is a newborn infant. In anadditional example, a subject is selected that is in need of inhibitingof an HIV infection. For example, the subject is either uninfected andat risk of HIV infection or is infected in need of treatment.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that expresses CD4 on its surface. Thesecells, also known as helper T cells, help orchestrate the immuneresponse, including antibody responses as well as killer T cellresponses. Th1 and Th2 cells are functional subsets of helper T cells.Th1 cells secrete a set of cytokines, including interferon-gamma, andwhose principal function is to stimulate phagocyte-mediated defenseagainst infections, especially related to intracellular microbes. Th2cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5,and whose principal functions are to stimulate IgE and eosinophillmastcell-mediated immune reactions and to downregulate Th1 responses.

Therapeutically effective amount: The amount of agent, such as adisclosed immunogen or immunogenic composition that is sufficient toprevent, treat (including prophylaxis), reduce and/or ameliorate thesymptoms and/or underlying causes of a disorder or disease, for exampleto prevent, inhibit, and/or treat HIV-1 infection. In some embodiments,a therapeutically effective amount is sufficient to reduce or eliminatea symptom of a disease, such as HIV-1 infection. For instance, this canbe the amount necessary to inhibit or prevent viral replication or tomeasurably alter outward symptoms of the viral infection. In general,this amount will be sufficient to measurably inhibit virus replicationor infectivity.

In one example, a desired response is to inhibit or reduce or preventHWV infection. The HIV infected cells do not need to be completelyeliminated or reduced or prevented for the composition to be effective.For example, administration of a therapeutically effective amount of theagent can decrease the number of HWV infected cells (or prevent theinfection of cells) by a desired amount, for example by at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or even at least 100% (elimination or prevention ofdetectable HWV infected cells), as compared to the number of HIVinfected cells in the absence of the composition.

It is understood that to obtain a protective immune response against apathogen can require multiple administrations of the immunogeniccomposition. Thus, a therapeutically effective amount encompasses afractional dose that contributes in combination with previous orsubsequent administrations to attaining a protective immune response.For example, a therapeutically effective amount of an agent can beadministered in a single dose, or in several doses, for example daily,during a course of treatment (such as a prime-boost vaccinationtreatment). However, the therapeutically effective amount can depend onthe subject being treated, the severity and type of the condition beingtreated, and the manner of administration. A unit dosage form of theagent can be packaged in a therapeutic amount, or in multiples of thetherapeutic amount, for example, in a vial (e.g., with a pierceable lid)or syringe having sterile components.

Transmembrane domain: An amino acid sequence that inserts into a lipidbilayer, such as the lipid bilayer of a cell or virus or virus-likeparticle. A transmembrane domain can be used to anchor an antigen to amembrane. In some examples a transmembrane domain is a HIV-1 Envtransmembrane domain. Exemplary HIV-1 Env transmembrane domains arefamiliar to the person of ordinary skill in the art, and providedherein, for example as SEQ ID NOs: 758, 760, and 762.

Treating or preventing a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as HIV-1 infection or acquired immunodeficiency syndrome(AIDS). “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. The term “ameliorating,” with referenceto a disease or pathological condition, refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject, a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease, areduction in the viral load, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease. A “prophylactic” treatmentis a treatment administered to a subject who does not exhibit signs of adisease or exhibits only early signs for the purpose of decreasing therisk of developing pathology.

The term “reduces” is a relative term, such that an agent reduces aresponse or condition if the response or condition is quantitativelydiminished following administration of the agent, or if it is diminishedfollowing administration of the agent, as compared to a reference agent.Similarly, the term “prevents” does not necessarily mean that an agentcompletely eliminates the response or condition, so long as at least onecharacteristic of the response or condition is eliminated. Thus, animmunogenic composition that reduces or prevents an infection or aresponse, can, but does not necessarily completely, eliminate such aninfection or response, so long as the infection or response ismeasurably diminished, for example, by at least about 50%, such as by atleast about 70%, or about 80%, or even by about 90% of (that is to 10%or less than) the infection or response in the absence of the agent, orin comparison to a reference agent.

Tyrosine Sulfation: Addition of a sulfate group to a tyrosine residue ina protein. In cells, tyrosine sulfation is a post translationalmodification where a sulfate group is added to a tyrosine residue of aprotein molecule in the Golgi or endoplasmic reticulum. Tyrosinesulfation can be catalyzed by a tyrosyl-protein sulfotransferase (TPST),such as TPST1 or TPST2. The reaction catalyzed by TPST is a transfer ofsulfate from the universal sulfate donor3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the side-chain hydroxylgroup of a tyrosine residue. Tyrosine sulfation can also be accomplishedin vitro, for example by incubating a peptide containing one or moretyrosine residues with a TBST enzyme (such as TBST1 or TBST2) underappropriate conditions. Methods of sulfating a tyrosine residue on aprotein are known (see, e.g., U.S. Pub. No. 5,541,095, 2009/0042738,2006/0009631, 2003/0170849, 2006/0115859, Liu et al., Mol. Biosyst.,7:38-47, 2011, and Choe and Farzan, Methods in Enzymology, 461: 147-170,2009) each of which is incorporated by reference herein).

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity.

Vaccine: A pharmaceutical composition that elicits a prophylactic ortherapeutic immune response in a subject. In some cases, the immuneresponse is a protective immune response. Typically, a vaccine elicitsan antigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell or one or more cellularconstituents. In one specific, non-limiting example, a vaccine reducesthe severity of the symptoms associated with HIV infection and/ordecreases the viral load compared to a control. In another non-limitingexample, a vaccine reduces HIV-1 infection compared to a control.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant nucleicacid vectors having at least some nucleic acid sequences derived fromone or more viruses. A replication deficient viral vector is a vectorthat requires complementation of one or more regions of the viral genomerequired for replication due to a deficiency in at least onereplication-essential gene function. For example, such that the viralvector does not replicate in typical host cells, especially those in ahuman patient that could be infected by the viral vector in the courseof a therapeutic method.

Virus-like particle (VLP): A non-replicating, viral shell, derived fromany of several viruses. VLPs are generally composed of one or more viralproteins, such as, but not limited to, those proteins referred to ascapsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. Further, VLPs can be isolated by known techniques, e.g.,density gradient centrifugation and identified by characteristic densitybanding. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456;and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J InvertebrPathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol,354: 53073, 2012).

VRC01: A neutralizing monoclonal antibody that specifically binds to theCD4 binding site on HIV-1 Env and can inhibit HIV-1 infection of targetcells. The person of ordinary skill in the art is familiar with theVRC01 mAb and with methods of its use and production (see, for example,Wu et al., Science, 329(5993):856-861, 2010, and PCT publicationWO2012/154312, each of which is incorporated by reference herein). Theamino acid sequences of the heavy and light variable regions of theVRC01 mAb are known and have been deposited in GenBank as Nos.ADF47181.1 (VRC01 V_(H)) and ADF47184.1 (VRC01 V_(L)), each of which isincorporated by reference herein as present in the database on Jun. 20,2014)

VRC26: A neutralizing monoclonal antibody that specifically binds to theV1/V2 domain of HIV-1 Env trimer in its prefusion mature closedconformation, and which can inhibit HIV-1 infection of target cells. Asused herein, “VRC26” refers to the VRC26.09 antibody, which is one ofseveral clonal variants isolated from donor CAP256. The person ofordinary skill in the art is familiar with the VRC26.09 mAb and withmethods of its use and production (see, for example, Doria-Rose et al.,Nature, 509, 55-62, 2014, which is incorporated by reference herein).The amino acid sequences of the heavy and light variable regions of theVRC26 mAb are known and have been deposited in GenBank as Nos. KJ134874(VRC26.09 V_(H)) and KJ134886 (VRC26.09 V_(L)), each of which isincorporated by reference herein as present in the database on Jun. 20,2014).

II. Description of Several Embodiments A. Native HIV-1 Sequences

HIV can be classified into four groups: the “major” group M, the“outlier” group O, group N, and group P. Within group M, there areseveral genetically distinct clades (or subtypes) of HIV-1. Thedisclosed recombinant HIV-1 Env proteins can be derived from any type ofHIV, such as groups M, N, O, or P, or clade, such as clade A, B, C, D,F, G, H, J, or K, and the like. HIV-1 Env proteins from the differentHIV clades, as well as nucleic acid sequences encoding such proteins andmethods for the manipulation and insertion of such nucleic acidsequences into vectors, are known (see, e.g., HIV Sequence Compendium,Division of AIDS, National Institute of Allergy and Infectious Diseases(2013); HIV Sequence Database(hiv-web.lanl.gov/content/hiv-db/mainpage.html); see, e.g., Sambrook etal. (Molecular Cloning: A Laboratory Manual, 4^(th) ed, Cold SpringHarbor, N.Y., 2012) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, through supplement 104,2013). Exemplary native HIV-1 Env protein sequences are available in theHIV Sequence Database (hiv-web.lanl.gov/content/hiv-db/mainpage.html),further, Table 5 provides sequences for Exemplary native HIV-1 Envproteins.

TABLE 5 Exemplary Native HIV-1 Env sequences Strain Clade Env sequenceHXB2 B SEQ ID NO: 1 BG505 A SEQ ID NO: 2 CAP256.SU C SEQ ID NO: 51BB201.B42 A SEQ ID NO: 81 KER2018.11 A SEQ ID NO: 107 CH070.1 BC SEQ IDNO: 174 ZM233.6 C SEQ ID NO: 745 Q23.17 A SEQ ID NO: 746 A244 AE SEQ IDNO: 747 WITO.33 B SEQ ID NO: 748 ZM53.12 C SEQ ID NO: 749 CNE58 C SEQ IDNO: 750 3301_V1_C24 AC SEQ ID NO: 751 T250-4 AE SEQ ID NO: 2114 JRFL BSEQ ID NO: 2115

In several embodiments, a disclosed immunogen can include a modification(e.g., cysteine substitutions that can form a disulfide bond tostabilize the HIV1 Env protein in a prefusion closed matureconformation) from a native HIV-1 Env protein sequence that has beendetermined to produce broadly neutralizing antibodies in a humansubject, for example broadly neutralizing antibodies that specificallybind the V1V2 domain of HIV-1 Env. For example, in some embodiments, anHIV-1 Env (or fragment thereof) sequence from a CAP256.SU, BB201.B42,KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, or WITO.33 strain ofHIV-1 is mutated to include one or more of the disclosed amino acidsubstitutions to generate a recombinant HIV Env protein (or fragmentthereof, such as a gp140 or gp145 protein) that is stabilized in aprefusion mature closed conformation. For example, in some non-limitingembodiments, cysteine substitutions at positions 201 and 433, and theSOSIP mutations, are made to a gp140 sequence from a CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, orWITO.33 strain of HIV-1 to generate the recombinant HIV-1 Env ectodomainthat can form a trimer stabilized in the prefusion mature closedconformation.

In view of the conservation and breadth of knowledge of HIV-1 Envsequences, the person of ordinary skill in the art can easily identifycorresponding HIV-1 Env amino acid positions between different HIV-1 Envstrains and subtypes. The HXB2 numbering system has been developed toassist comparison between different HIV amino acid and nucleic acidsequences. The person of ordinary skill in the art is familiar with theHXB2 numbering system (see, e.g., Korber et al, Human Retroviruses andAIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino AcidSequences. Korber B, Kuiken C L, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, LosAlamos National Laboratory, Los Alamos, N. Mex., which is incorporatedby reference herein in its entirety). The numbering of amino acidsubstitutions disclosed herein is made according to the HXB2 numberingsystem, unless context indicates otherwise.

B. Recombinant HIV-1 Env Ectodomains Stabilized in a Prefusion MatureClosed Conformation

Isolated immunogens are disclosed herein that include a recombinantHIV-1 Env ectodomain trimer or immunogenic fragment thereof that ismodified from a native form (e.g., by introduction of one or more aminoacid substitutions) to be stabilized in the prefusion mature closedconformation.

The HIV-1 Env ectodomain trimer can include a prefusion mature closedconformation wherein the V1V2 domain of each Env ectodomain protomer inthe trimer comes together at the membrane distal apex. At the membraneproximal aspect, the HIV-1 Env ectodomain trimer in the prefusion matureclosed conformation includes distinct α6 and α7 helices; the α7 helixdoes not start until after residue 570. For example, in the prefusionmature closed conformation, the interprotomer distance between residues200 and 313 can be less than 5 Angstroms.

In several embodiments, the immunogen includes a recombinant HIV-1 Envectodomain trimer, which can include, for example, a trimeric complex ofrecombinant HIV-1 Env ectodomains that are stabilized in the prefusionmature closed conformation by one or more amino acid substitutions. Therecombinant HIV-1 Env ectodomain trimer typically includes a proteincomplex of gp120-gp41 ectodomain protomers. The gp120-gp41 protomer caninclude separate gp120 and gp41 polypeptide chains, or can include gp120and gp41 polypeptide chains that are linked (e.g., by a peptide linker)to form a single polypeptide chain (e.g., as described in the “singlechain” section below). In several embodiments, the recombinant HIV-1 Envectodomain trimer is membrane anchored and can include a trimericcomplex of recombinant HIV-1 Env ectodomains that are linked to atransmembrane domain (e.g., a gp145 protein including a gp120 proteinand a gp41 ectodomain and transmembrane domain).

The recombinant HIV-1 Env ectodomain includes a gp120 protein and a gp41ectodomain. The gp120 protein typically does not include a signalpeptide (for example, the gp120 protein typically does not include gp120residues 1-30), as the signal peptide is proteolytically cleaved duringcellular processing. Additionally, the gp41 ectodomain includes theextracellular portion of gp41 (e.g., positions 512-664). In embodimentsincluding a soluble recombinant HIV-1 Env ectodomain, the gp41ectodomain is not linked to a transmembrane domain or other membraneanchor. However, in embodiments including a membrane anchoredrecombinant HIV-1 Env ectodomain the gp41 ectodomain can be linked to atransmembrane domain (such as, but not limited to, an HIV-1 Envtransmembrane domain).

In several embodiments, the recombinant HIV-1 Env ectodomain includes agp120 polypeptide and a gp41 ectodomain, wherein

the n-terminal residue of the gp120 polypeptide is one of HIV-1 Envpositions 1-35;

the c-terminal residue of the gp120 polypeptide is one of HIV-1 Envpositions 503-511;

the n-terminal residue of the gp41 ectodomain is one of HIV-1 Envpositions 512-522; and/or

the c-terminal residue of the gp41 ectodomain is one of HIV-1 Envpositions 624-705.

In one non-limiting example, the recombinant HIV-1 Env ectodomainincludes a gp120 polypeptide and a gp41 ectodomain, wherein then-terminal residue of the gp120 polypeptide is HIV-1 Env position 31;the c-terminal residue of the gp120 polypeptide is HIV-1 Env position511; the n-terminal residue of the gp41 polypeptide is HIV-1 Envposition 512; and/or the c-terminal residue of the gp41 polypeptide isHIV-1 Env position 664. In some embodiments, the C-terminal residue ofthe recombinant HIV-1 Env ectodomain is position 683 (the entireectodomain, terminating just before the transmembrane domain). Inadditional embodiments, the C-terminal residue of the recombinant HIV-1Env ectodomain is position 707 (the entire ectodomain, terminating justafter the transmembrane domain).

Native HIV-1 Env sequences include a furin cleavage site (e.g., REKR,SEQ ID NO: 572) between positions 508 and 512 (HXB2 numbering), thatseparates gp120 and gp41. Any of the disclosed recombinant HIV-1 Envectodomains can further include an enhanced cleavage site between gp120and gp41 proteins. The enhanced cleavage cite can include, for example,substitution of six arginine resides for the four residues of the nativecleavage site (e.g., REKR (SEQ ID NO: 572) to RRRRRR (SEQ ID NO: 573).As used herein, reference to “R6” indicates that a HIV Env proteinincludes the RRRRRR (SEQ ID NO: 573) substitution for the native furincleavage site. It will be understood that protease cleavage of the furinor enhanced cleavage site separating gp120 and gp41 can remove a fewamino acids from either end of the cleavage site.

Stabilization of the recombinant HIV-1 Env ectodomain trimer orimmunogenic fragment in the prefusion mature closed conformationprevents transition of the HIV-1 Env ectodomain to the CD-bound openconformation. Thus, the disclosed recombinant HIV-1 Env ectodomaintrimers can be specifically bound by an antibody that is specific forthe mature closed conformation of HIV-1 Env (e.g., VRC26, PGT151,PGT122, or PGT145), but are not specifically bound by an antibodyspecific for the CD4-bound open conformation, of HIV-1 Env (e.g., 17 bmAb in the presence of sCD4). In one example, the recombinant HIV-1 Envectodomain trimer is not specifically bound by an antibody specific fora CD4-induced epitope on the recombinant HIV-1 Env ectodomain trimer,such as the 17b antibody. Methods of determining if a recombinant HIV-1Env ectodomain trimer includes a CD4-induced epitope are known in theart and disclosed herein (See Examples 1 and 2). For example, theantibody binding assay can be conducted in the presence of a molarexcess of soluble CD4 as described in Sanders et al. (Plos Pathogens, 9,e1003618, 2013).

In several embodiments, the recombinant HIV-1 Env ectodomain trimers canbe specifically bound by an antibody that specifically binds to the V1V2domain on a HIV-1 Env trimer, but not an Env monomer. Exemplaryantibodies with such antigen binding characteristics include the PGT141,PGT142, PGT143, PGT144, PGT145, and VRC26 antibodies. Additionalexamples include the PG9, PG16, and CH01-CH04 antibodies. Accordingly,in some embodiments the recombinant HIV-1 Env ectodomain trimerspecifically binds to an antibody (such as a PGT141, PGT142, PGT143,PGT144, PGT145, and VRC26 antibody) that specifically binds to the V1V2domain of a HIV-1 Env in its trimeric, but not monomeric, form with adissociation constant of less than 10⁻⁶ Molar, such as less than 10⁻⁷Molar, less than 10⁻⁸ Molar, or less than 10⁻⁹ Molar. Specific bindingcan be determined by methods known in the art. The determination ofspecific binding may readily be made by using or adapting routineprocedures, such as ELISA, immunocompetition, surface plasmon resonance,or other immunosorbant assays (described in many standard texts,including Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL,New York, 1999).

The recombinant HIV-1 Env ectodomain trimers or immunogenic fragmentsare stabilized in the prefusion mature closed conformation by one ormore amino acid substitutions. Thus, the recombinant HIV-1 Envectodomain trimers or immunogenic fragments are not stabilized bynon-specific crosslinking, for example glutaraldehyde crosslinking ofmembrane bound HIV-1 Env trimers.

In several embodiments, the recombinant HIV-1 Env ectodomain trimer issoluble in aqueous solution. In some embodiments, the recombinant HIV-1Env ectodomain trimer dissolves to a concentration of at least 0.5 mg/ml(such as at least 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/mlor at least 5.0 mg/ml) in phosphate buffered saline (pH 7.4) at roomtemperature (e.g., 20-22 degrees Celsius) and remains dissolved for atleast for at least 12 hours (such as at least 24 hours, at least 48hours, at least one week, at least two weeks, or more time). In oneembodiment, the phosphate buffered saline includes NaCl (137 mM), KCl(2.7 mM), Na₂HPO₄ (10 mM), KH₂PO₄ (1.8 mM) at pH 7.4. In someembodiments, the phosphate buffered saline further includes CaCl₂ (1 mM)and MgCl₂ (0.5 mM). The person of skill in the art is familiar withmethods of determining if a protein remains in solution over time. Forexample, the concentration of the protein dissolved in an aqueoussolution can be tested over time using standard methods.

In some embodiments, the recombinant HIV-1 Env ectodomain trimer, whenincubated in an aqueous solution, forms a population of recombinantHIV-1 Env ectodomain trimers stabilized in a prefusion mature closedconformation, wherein at least 70% (such as at least 80%, or at least90% or at least 95% or at least 98%) of the recombinant HIV-1 Envectodomain trimers in the population specifically bind to an antibodythat specifically binds to the V1V2 domain of a trimer, but notmonomeric HIV-1 Env (such as a PGT141, PGT142, PGT143, PGT144, PGT145,and VRC26 antibody) after

(a) incubation for one hour in 350 mM NaCl pH 7.0, at 50° C.;

(b) incubation for one hour in 350 mM NaCl pH 3.5, at 25° C.;

(c) incubation for one hour in 350 mM NaCl pH 10, at 25° C.;

(d) incubation for one hour in 10 mM osmolarity, pH 7.0, at 25° C.;

(e) incubation for one hour in 3000 mM osmolarity, pH 7.0, at 25° C.;

(g) a combination of two or more of (a)-(e); or

a combination of (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b)and (d); (b) and (e); (c) and (d); (c) and (e); (a), (b), and (d); (a),(c), and (d); (a), (b), and (e); or (a), (c), and (e).

In some embodiments, the recombinant HIV-1 Env ectodomain trimer, whenincubated in an aqueous solution, forms a population of recombinantHIV-1 Env ectodomain trimers stabilized in a prefusion mature closedconformation, wherein at least 70% (such as at least 80%, or at least90% or at least 95% or at least 98%) of the recombinant HIV-1 Envectodomain trimers in the population specifically bind to an antibodythat specifically binds to the V1V2 domain of a trimer, but notmonomeric HIV-1 Env (such as a PGT141, PGT142, PGT143, PGT144, PGT145,and VRC26 antibody) ten freeze-thaw cycles in 350 mM NaCl pH 7.0.

Several embodiments include a multimer of the recombinant HIV-1 Envectodomain trimer or immunogenic fragment thereof, for example, amultimer including 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more of therecombinant HIV-1 Env ectodomain trimers or immunogenic fragmentthereof.

It is understood in the art that some variations can be made in theamino acid sequence of a protein without affecting the activity of theprotein. Such variations include insertion of amino acid residues,deletions of amino acid residues, and substitutions of amino acidresidues. These variations in sequence can be naturally occurringvariations or they can be engineered through the use of geneticengineering technique known to those skilled in the art. Examples ofsuch techniques are found in see, e.g., Sambrook et al. (MolecularCloning: A Laboratory Manual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012)and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, through supplement 104, 2013, both of which areincorporated herein by reference in their entirety.

The recombinant HIV-1 Env ectodomain can include modifications of thenative HIV-1 sequence, such as amino acid substitutions, deletions orinsertions, glycosylation and/or covalent linkage to unrelated proteins(e.g., a protein tag), as long as the recombinant HIV-1 Env ectodomaincan form a trimer that is stabilized in the prefusion mature closedconformation. HIV-1 Env proteins from the different Clades, as well asnucleic acid sequences encoding such proteins and methods for themanipulation and insertion of such nucleic acid sequences into vectors,are disclosed herein and known in the art.

In some embodiments a recombinant HWV-1 Env ectodomain included in thedisclosed trimers includes a gp120 polypeptide and a gp41 ectodomainincluding amino acid sequences at least 75% (for example at least 85%,90%, 95%, 96%, 97%, 98% or 99%) sequence identity to a correspondingnative HIV-1 gp120 or gp41 ectodomain polypeptide sequence (e.g., anative gp120 or gp41 ectodomain protein sequence from a clade A, B, C,D, F, G, H, J or K HIV-1 Env protein), such a native HIV-1 sequenceavailable in the HIV Sequence Database(hiv-web.lanl.gov/content/hiv-db/mainpage.html) or a native HIV-1 Envpolypeptide sequence set forth in Table 5, and include the one or moreamino acid substitutions that stabilize the protein in the prefusionmature closed conformation.

In additional embodiments, a recombinant HIV-1 Env ectodomain includedin the disclosed trimers includes a gp120 polypeptide and/or a gp41ectodomain including one or more amino acid substitutions compared to acorresponding native HIV-1 Env sequence. For example, in someembodiments, the gp120 polypeptide, gp41 ectodomain, or both, caninclude up to 20 (such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19) amino acid substitutions compared to anative HIV-1 gp140 polypeptide sequence (e.g., a native gp120 or gp41ectodomain protein sequence from a clade A, B, C, D, F, G, H, J or KHIV-1 Env protein), such a native HIV-1 sequence available in the HIVSequence Database (hiv-web.lanl.gov/content/hiv-db/mainpage.html) or anative HIV-1 Env polypeptide sequence set forth in Table 5, and includethe one or more amino acid substitutions that stabilize the protein inthe prefusion mature closed conformation. The simplest modificationsinvolve the substitution of one or more amino acids for amino acidshaving similar biochemical properties. These so-called conservativesubstitutions are likely to have minimal impact on the activity of theresultant protein.

The recombinant HIV-1 Env ectodomain included in the disclosed trimerscan be derivatized or linked to another molecule (such as anotherpeptide or protein). In general, the recombinant HIV-1 Env ectodomain isderivatized such that the binding to broadly neutralizing antibodies toa trimer of the recombinant HIV-1 ectodomain, such as PGT122, is notaffected adversely by the derivatization or labeling. For example, therecombinant HIV-1 Env ectodomain can be functionally linked (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as an antibody or protein ordetection tag.

The stabilizing modifications provided herein are targeted modificationsthat stabilize the recombinant HIV-1 Env ectodomain trimer in theprefusion mature closed conformation. Guided by the structural featuresidentified in the prefusion mature closed conformation, several modes ofstabilizing the HIV-1 Env ectodomain trimer in this conformation areavailable, including (but not limited to) amino acid substitutions thatintroduce one or more non-natural disulfide bonds, fill cavities withinthe HIV-1 ectodomain trimer, prevent structural rearrangements,introduce N-linked glycosylation sites, and combinations thereof.Corresponding mutations are discussed in more detail below:

Reference to Table 13

Several stabilizing mutations are provided that can be included on aHIV-1 ectodomain to generate a trimeric HIV-1 ectodomain stabilized inthe prefusion mature closed conformation. These mutations include, butare not limited to, those provided in Table 13. In several embodiments,the recombinant HIV-1 Env ectodomain trimer includes a HIV-1 Envectodomain including the amino acid substitutions set forth in any onerow of column 5, column 6, or columns 5 and 6, of Table 13. Inadditional embodiments, the recombinant HIV-1 Env ectodomain trimerincludes a HIV-1 Env ectodomain including an amino acid sequence atleast 80% identical to any one of the modified HIV-1 Env Ectodomainsequences listed in Table 13. In further embodiments, the recombinantHIV-1 Env ectodomain trimer includes a HIV-1 Env ectodomain includingthe amino acid sequence of any one of the modified HWV-1 Env Ectodomainsequences listed in of Table 13

Some of the sequences of recombinant HIV-1 ectodomains provided hereininclude the sequence of protease cleavage sites (such as thrombinsites), protein tags (such as a His tag, a Strep Tag II, a Avi tag,etc.), signal peptides, that the person of ordinary skill in the artwill understand would not be included in an isolated immunogen includinga recombinant HIV-1 Env ectodomain immunogen. The person of ordinaryskill in the art will recognize such sequences, and when appropriate,understand that these tags or protease cleavage sites are not includedin a disclosed recombinant HIV-1 Env protein.

In some embodiments, the recombinant HWV-1 Env ectodomain trimerincludes the SOSIP, R6, 664 and 201C/433C modifications, an asparagineat position 332, and the substitutions listed in column 6 of any one ofthe rows for SEQ ID NO: 1245-1367 or 1580-1610 of Table 13. In someembodiments, the recombinant HWV-1 Env ectodomain trimer includes theSOSIP, R6, and 664 substitutions, an asparagine at position 332, and thesubstitutions listed in column 6 of any one of the rows for SEQ ID NO:1368-1399 or 1580-1610 of Table 13. In several such embodiments, therecombinant HIV-1 Env ectodomain trimer can be based on a JRFL or aBG505 strain of HWV-1. In some embodiments, the gp120/gp41 ectodomainsin the recombinant HIV-1 Env ectodomain trimer can comprise an aminoacid sequence set forth as any one of SEQ ID NO: 1245-1399 or 1580-1610,or a sequence at least 90% identical thereto.

In some embodiments, the recombinant HWV-1 Env ectodomain trimerincludes the SOSIP, R6, 664, and 201C/433C substitutions, an asparagineat position 332, and is based on a strain of HIV-1 as listed in column 4of any one of the rows for SEQ ID NO: 26, 1057-1077, 1400-1579 of Table13. In some embodiments, the gp120/gp41 ectodomains in the recombinantHIV-1 Env ectodomain trimer can comprise an amino acid sequence setforth as any one of SEQ ID NO: 26, 1057-1077, 1400-1579, or a sequenceat least 90% identical thereto.

Non-Natural Disulfide Bonds

In several embodiments, the recombinant HIV-1 Env ectodomain trimerincludes one or more non-natural disulfide bonds that stabilize theHIV-1 Env ectodomain trimer in the prefusion mature closed conformation.A non-natural disulfide bond is one that does not occur in a nativeHIV-1 Env protein, and is introduced by protein engineering (e.g., byincluding one or more substituted cysteine residues that form thenon-natural disulfide bond). For example, in some embodiments, any ofthe disclosed recombinant HIV-1 Env ectodomain trimers can be stabilizedin a prefusion mature closed conformation by any one of 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 non-natural disulfide bonds.

The cysteine residues that form the disulfide bond can be introducedinto a native HIV-1 sequence by one or more amino acid substitutions.For example, in some embodiments, a single amino acid substitutionintroduces a cysteine that forms a disulfide bond with a cysteineresidue present in the native HIV-1 sequence. Alternately, two cysteineresidues can be introduced into a native HIV-1 Env ectodomain sequenceto form the disulfide bond. The location of the cysteine (or cysteines)of the non-natural disulfide bond can be determined by the person ofordinary skill in the art using the disclosed structure of the HIV-1ectodomain trimer in a prefusion mature closed conformation.

For example, the amino acid positions of the cysteines are typicallywithin a sufficiently close distance for formation of a disulfide bondin the prefusion mature closed conformation of the HIV-1 Env proteintrimer. Methods of using three-dimensional structure data to determineif two residues are within a sufficiently close distance to one anotherfor disulfide bond formation are known (see, e.g., Peterson et al.,Protein engineering, 12:535-548, 1999 and Dombkowski, Bioinformatics,19:1852-1853, 3002 (disclosing DISULFIDE BY DESIGN™), each of which isincorporated by reference herein). Residues can be selected manually,based on the three dimensional structure of the HIV-1 Env trimer in aprefusion mature closed conformation provided herein, or a software,such as DISULFIDEBYDESIGN™, can be used. Without being bound by theory,ideal distances for formation of a disulfide bond are generallyconsidered to be about ˜5.6 Å for Cα-Cα distance, ˜2.02 Å for Sγ-Sγdistance, and 3.5-4.25 Å for Cβ-Cβ distance (using the optimal rotomer).The person of ordinary skill in the art will appreciate that variationsfrom these distances are included when selecting residues in a threedimensional structure that can be substituted for cysteines forintroduction of a disulfide bond. For example, in some embodiments theselected residues have a Cα-Cα distance of less than 7.0 Å and/or aCβ-Cβ distance of less than 4.7 Å. In some embodiments the selectedresidues have a Cα-Cα distance of from 2.0-8.0 Å and/or a Cβ-Cβ distanceof from 2.0-5.5 Å. In several embodiments, the amino acid positions ofthe cysteines are within a sufficiently close distance for formation ofa disulfide bond in the prefusion mature closed conformation, but notthe CD4-bound open conformation of the HIV-1 Env protein.

For example, the person of ordinary skill in the art can determine therelative position of a particular amino acid between the prefusionmature closed and CD4-bound conformations of the HIV-1 Env ectodomain bycomparing the prefusion mature closed structures disclosed in theExamples and the structural coordinates provided in Tables 1-4, with thepreviously identified CD4-bound conformation described in Example 1.Methods of determining relative position of a particular amino acidbetween the two protein structures (e.g., between the three dimensionalstructures prefusion mature closed- and CD4-bound-HIV-1 Env protein) areknown. For example the person of ordinary skill in the art can use knownsuperimposition methods to compare the two structures (e.g., methodsusing the LSQKAB program (Kabsch W. Acta. Cryst. A32 922-923 (1976)).

In several embodiments, the recombinant HIV-1 Env protein is stabilizedin a prefusion mature closed conformation by a disulfide bond between acysteine introduced at an amino acid position that changes conformation,and a cysteine introduced into an amino acid position that does notchange conformation, between the prefusion mature closed conformationand the CD4-bound conformation of HIV-1 Env. For example, in someembodiments, the recombinant HIV-1 Env protein is stabilized in aprefusion mature closed conformation by a disulfide bond between a pairof cysteines, wherein the first cysteine is in an amino acid position ofthe HIV-1 Env protein that has a root mean square deviation of at least5 (such as at least 6, at least 7, at least 8, at least 9 or at least10) angstroms between the three-dimensional structure of the HIV-1 Envprotein prefusion mature closed and CD4-bound conformations, and thesecond cysteine is in an amino acid position of the HIV-1 Env proteinthat has a root mean square deviation of less than 4 (such as less than3, 2, or 1) angstroms between the three-dimensional structure of theHIV-1 Env protein prefusion mature closed and CD4-bound conformations.

In additional embodiments, the recombinant HIV-1 Env protein isstabilized in a prefusion mature closed conformation by a disulfide bondbetween cysteines that are introduced at amino acid positions that bothchange conformation between the prefusion mature closed and CD4-boundconformations. For example, in some embodiments, the recombinant HIV-1Env protein includes amino acid substitutions introducing a pair ofcysteines, wherein the first cysteine and the second cysteine are atamino acid positions of the HIV-1 Env protein that both have a root meansquare deviation of at least 5 (such as at least 6, at least 7, at least8, at least 9 or at least 10) angstroms between the three-dimensionalstructure of the prefusion mature closed and CD4-bound conformations ofthe HIV-1 Env protein.

In several embodiments the recombinant HIV-1 Env ectodomain included inthe trimer includes one or more amino acid substitutions that stabilizethe V1V2 domain “cap” and/or V3 domain in the prefusion mature closedconformation.

For example, in some embodiments, the recombinant HIV-1 Env ectodomainincluded in the trimer includes a non-natural disulfide bond between afirst cysteine in a position of the β2 sheet and a second cysteine in agp120 positions of the β21 sheet of the HIV-1 ectodomain in the matureclosed conformation as disclosed herein (see FIG. 11 ).

In some embodiments, the gp120 polypeptide in the recombinant HIV-1 Envectodomain can include a non-natural disulfide bond between a pair ofcysteine substitutions at one of gp120 positions 179-180 and one ofgp120 positions 420-423; one of gp120 positions 190-210 and one of gp120positions 425-437; one of gp120 positions 198-202 and one of gp120positions 428-437; one of gp120 positions 179-180 and one of gp120positions 421-423; or one of gp120 positions 195-201 to one of gp120positions 423-433, wherein the non-natural disulfide bond stabilizes theHIV-1 Env ectodomain in the prefusion mature closed conformation.

In some embodiments, the gp120 polypeptide in the recombinant HIV-1 Envectodomain can include a pair of cysteine substitutions that can form adisulfide bond to stabilize a trimer of the recombinant HIV-1 Envectodomain in the mature closed conformation. Exemplary gp120 positionsthat can be mutated to cysteine, as well as exemplary mutations (in thecontext of the BG505 swrain), and an exemplary sequence including theindicated mutations are provided in Table 6.

TABLE 6 Non-natural gp120-gp120 disulfide bonds. Exemplary Env Exemplarypositions Substitutions Intra- or (HXB2 (HXB2 Inter- Exemplarynumbering) numbering) protomer? Comment SEQ ID NO  36 and 496 V36C/V496CIntra Stabilize gp120 N and C termini 649  36 and 498 V36C/P498C IntraStabilize gp120 N and C termini 650  37 and 497 T37C/A497C IntraStabilize gp120 N and C termini 651  38 and 496 V38C/V496C IntraStabilize gp120 N and C termini 652  36 and 608 V36C/V608C Intrastabilize V1V2 mature closed conformation 82 55 and 77 A55C/T77C IntraInhibit α0 formation 683 57 and 77 D57C/T77C Intra Inhibit α0 formation98 58 and 77 A58C/T77C Intra Inhibit α0 formation 97  66 and 209V66C/S209C Intra Inhibit α0 formation 101  68 and 208 V68C/V208C IntraInhibit α0 formation 100  68 and 209 V68C/S209C Intra Inhibit α0formation 99 120 and 315 V120C/Q315C Intra stabilize V1V2 mature closedconformation 70 122 and 125 L122C/L125C Intra stabilize V1V2 matureclosed conformation 64 122 and 203 L122C/Q203C Intra stabilize V1V2mature closed conformation 77, 785 122 and 317 L122C/F317C Intra Lockingthe V3 to gp120 to prevent exposure/opening 789 122 and 433 L122C/A433CIntra stabilize V1V2 mature closed conformation 162 124 and 164P124C/T164C Inter stabilize V1V2 mature closed conformation 71 124 and166 P124C/R166C Inter stabilize V1V2 mature closed conformation 20 128and 165 T128C/L165C Inter stabilize V1V2 mature closed conformation 128and 167 T128C/T167C Inter stabilize V1V2 mature closed conformation 72163 and 170 T163C/Q170C Intra Stabilize V1V2 662 164 and 197 E164C/N197CInter stabilize V1V2 mature closed conformation 19 164 and 308S164C/H308C Intra Locking the V3 to V1V2 to prevent exposure/opening 794172 and 307 E172C/I307C Intra Locking the V3 to V1V2 to preventexposure/opening 791 174 and 318 S174C/A319C Intra stabilize V1V2 matureclosed conformation 8 174 and 319 S174C/T319C Intra Locking the V3 toV1V2 to prevent exposure/opening 793 175 and 320 L175C/T320C Intrastabilize V1V2 mature closed conformation  9, 795 176 and 180F176C/D180C Intra stabilize V1V2 mature closed conformation 57 180 and421 D180C/K421C Intra stabilize V1V2 mature closed conformation 28 180and 423 D180C/I423C Intra stabilize V1V2 mature closed conformation 21195 and 423 N195C/I423C Intra stabilize V1V2 mature closed conformation22 195 and 433 N195C/A433C Intra stabilize V1V2 mature closedconformation 23, 777 199 and 431 S199C/G431C Intra stabilize V1V2 matureclosed conformation 25 199 and 433 S199C/A433C Intra stabilize V1V2mature closed conformation 24, 778 200 and 313 A200C/P313C Interstabilize V1V2 mature closed conformation 12 200 and 432 A200C/Q432CIntra Stabilize β21 to V1V2 653 201 and 433 I201C/A433C Intra stabilizeV1V2 mature closed conformation 26, 773 202 and 434 T202C/M434C IntraStabilize β21 to V1V2 654 202 and 433 T202C/A433C Intra Stabilize β21 toV1V2 656 203 and 317 Q203C/F317C Intra Locking the V3 to gp120 toprevent exposure/opening 788 204 and 434 A204C/M434C Intra stabilizeV1V2 mature closed conformation 63 204 and 436 A204C/A436C Intrastabilize V1V2 mature closed conformation 58 206 and 318 P206C/Y318CIntra Locking the V3 to gp120 to prevent exposure/opening 792 212 and252 P212C/K252C intra stabilize V1V2 mature closed conformation 60 225and 245 I225C/V245C Intra Stabilizes V1V1 646 225 and 488 I225C/V488CIntra Stabilize V1V2 mature closed conformation 659 257 and 375T257C/S375C Intra Stabilize V1V2 mature closed conformation 682 294 and333 I294C/V333C Intra Stabilize core 664 298 and 329 R298C/A329C Intrastabilize V1V2 mature closed conformation 74 304 and 440 R304C/Q440CIntra Stabilize prefusion close conformation (through V3) 743, 779, 787318 and 437 Y318C/P437C Intra stabilize V1V2 mature closed conformation53, 790 320 and 438 T320C/P438C Intra stabilize V1V2 mature closedconformation 27, 796 364 and 372 S364C/T372C Intra stabilize V1V2 matureclosed conformation 80 370 and 426 E370C/M426C Intra stabilize strandβ20 to core gp120 170 380 and 426 G380C/M426C Intra stabilize strand β20to core gp120 171 382 and 424 F382C/I424C Intra stabilize V1V2 matureclosed conformation 73 425 and 433 N425C/A433C Intra stabilize V1V2mature closed conformation (stabilize 69, 783 β20 to β21) 425 and 430N425C/I430C Intra Stabilizes CD4 binding loop (stabilize β20 to β21) 52

In one non-limiting embodiment, the recombinant HIV-1 Env ectodomain caninclude cysteine substitutions at positions 201 and 433 (e.g., I201C andA433C substitutions). In additional embodiments, the recombinant HIV-1Env ectodomain can include cysteine substitutions at positions 201 and433 (e.g., I201C and A433C substitutions) and further include one ormore additional mutations as disclosed herein. Exemplary additionalmutations include those disclosed herein that stabilize the V1V2 domain,the V3 domain, or the CD4 binding site in the prefusion mature closedconformation, or a mutation that stabilizes gp41 in the prefusion matureclosed conformation, such as a mutation that inhibits HR1 or HR2formation or fusion peptide extension. Non-limiting examples ofmutations that can be combined with the cysteine substitutions atpositions 201 and 433 include the SOS, IP, SOSIP mutations, as well asany of the mutations listed in Table 9, below.

Cavity Filling Amino Acid Substitutions

Comparison of the structure of the mature closed conformation of theHIV-1 Env ectodomain trimer (e.g., in complex with PGT122 and 35O22 Fabsas disclosed herein) to the structure of the CD4-bound conformation ofHIV-9 Env identifies several internal cavities or pockets in the matureclosed conformation that collapse when the HIV-1 Env ectodomain trimertransitions from the prefusion closed conformation to the CD4-bound openconformation. These cavities include those listed in Table 8, below.

Accordingly, in several embodiments, the recombinant HIV-1 Envectodomain trimer can be stabilized in the mature closed conformation byone or more amino acid substitutions that reduce the volume of aninternal cavity that collapses in the CD4-bound conformation of theHIV-1 Env ectodomain trimer. For example, cavities can be filled bysubstituting amino acids with large side chains for those with smallside chains. The cavities can be intra-protomer cavities, orinter-protomer cavities. The person of ordinary skill in the art can usemethods provided herein to compare the structures of the mature closedand CD34-bound conformations of HIV-1 Env to identify suitable cavities,and amino acid substitutions for filling the identified cavities.Exemplary cavities, amino acid substitutions for reducing the volume ofthese cavities are provided in Table 8.

Exemplary HIV-1 Env positions for introducing cavity filling amino acidsubstitutions that stabilize the prefusion mature closed conformation ofthe HIV-1 Env ectodomain trimer are provided in Table 8, as arecorresponding amino acid substitutions with reference to the BG505strain, and an exemplary sequence including the indicated substitution.

TABLE 8 Exemplarity cavity-filling amino acid substitutions Exemplarysubstitution Exemplary Exemplary Position residues substitution(s) SEQID NO Cavity Location (CL) and stabilizing mechanism (SM) 39 W, M, I, FY39F, Y39W 47-48 CL: gp120/gp41 interface SM: fill cavity and addhydrophobic interactions at the gp120- gp41 interactive surface 50 F, Y,L, I, M, V, W T50W 181 CL: gp120/gp41 interface SM: stabilize gp120/gp41interface 53 W F53W 197 CL: gp120-gp41 interface, near N-term of β-2 SM:Fill cavity between gp120 and gp41 to stabilize gp41 disordered regionand gp120/gp41 interaction 55 F A55F 678 CL: gp120 C1 with Fsubstitution to stabilize gp120 N terminus 61 W Y61W 195 CL: Middle ofα1 SM: Fill cavity between gp120 and gp41; prevent CD4-induced α0formation/α1 disruption 68 F, W, Y, L, I, M V68L 196 CL: Loop betweenα-1 and β0 SM: Fill cavities that are otherwise filled by CD4 induced α0formation 70 F, Y, W A70F, A70Y 143, 144 CL: Close to A70, α0/α7 SM:Extension of hydrophobic/aromatic patch-gp41/gp120 stabilization 75 W,F, M V75W, V75F, 90, 91, CL: gp120/gp41 interface V75M 92 SM: stabilizegp120/gp41 interface 77 F T77F 676 CL: gp120 C1 with F substitution tostabilize gp120 N terminus 110 F, Y, L, I, M, V, W S110W 178 CL: trimeraxis/gp41 interface boundary SM: stabilize prefusion axisinteractions/gp41 interface/prevent helix movement 111 F, Y, W L111Y,L111F, 145, 146, CL: gp120 C1, Close to A70, α0/α7 L111W 697 SM:Extension of hydrophobic/aromatic patch-gp41/gp120 stabilization (α7/α0,cavity close to A70), Substitute W to stabilize gp120 terminus 114 F, Y,L, I, M, V, W Q114W 179 CL: trimer axis/gp41 interface boundary SM:stabilize prefusion axis interactions/gp41 interface/prevent helixmovement 115 F, W, Y, L, I, M, V S115W 139 CL: C-term of α1 SM: Reducescavity at top of α1, which shifts conformation in CD4-bound state 117 F,Y, L, I, H, R, K117E, K117W 175, 176 CL: trimer axis E, D, M, V, W SM:stabilize prefusion axis interactions 118 F, W, Y, L, I, M, V P118W 140CL: SM: Reduces cavity near top (c-term) of α1, which shiftsconformation in CD4-bound state 120 F, Y, L, I, H, R, E, V120W 128 CL:Under V1V2 cap at N-term of β2 D, M, V, W SM: stabilize V1V2 matureclosed conformation/prevent bridging sheet formation, β2 extends inCD4-bound state, this stabilizes small hydrophobic pocket in groundstate 121 F, Y, L, I, H, R, E, K121E 177 CL: trimer axis D, M, V SM:stabilize prefusion axis interactions 123 F, Y, L, M, V, W T123W 172 CL:trimer axis SM: stabilize prefusion axis interactions 125 F, W, Y, L, I,M, V L125W, L125F 131, 685 CL: Cavity near N-term of V1V2 domain and V3near residue 127 and 126-196 disulfide of V1V2 SM: Removal of groundstate destabilization/flexibility cavity filling, stabilize V1/V2/V3 136W N136W 686 CL: gp120 V1. Substitute W to stabilize V1/V2/V3 139 F, M,I, Y, T; T139W  45 CL: interface of V1V2 and V3 loops SM: Stabilizeinteractions between V1V2 and V3 loops in the mature closed state 151 F,W, Y, L, I, M, V R151E, 132 CL: near V1 loop at position 153 SM: Addinghydrophobic patch at V1 loop to V3 loop 153 F, W E153F, E153W 707, 708CL: V1V2-V3/gp120core interface, 154 F, W L154F, L154W 709, 710 CL:gp120 V1/V2. Substitute F, Y or W, stabilize V1/V2/V3 159 W, Y F159W,F159Y 133, 30  CL: Primary hydrophobic pocket between V1V2 and V3 nearresidue 159 SM: Stabilize hydrophobic core of V1V2-V3 interactions 161F, W, Y, L, I M161W 135 CL: Hydrophobic patch at Cterm of V1V2 strand BSM: Stabilize V1V2 strand B to V3 near trimeric interface 164 F, WE164F, E164W 711, 712 CL: inter-protomer, Substitute F or W, 173 Y173W703 CL: gp120 V1/V2/V3. 173: Substitute W or F 175 F, W L175F, L175W705, 706 CL: V1V2-V3 interface, Substitute F or W, 176 W F176W 733 CL:gp120 V1/V2. Substitute Y or W, stabilize V1/V2/V3 177 W Y177W 198 CL:C-term of beta C SM: Fills cavity between V3 and gp120 core to stabilizeclosed V1V2 cap 179 F, Y, M, I, W L179W  46 CL: V1V2 loop gp120 coreinterface SM: Stabilize V1V2 loop-gp120 core interaction in matureclosed state 180 180: L, V, I, M D180L 123 CL: V1V2 interaction nearresidue 180 with V3 and base of β21 SM: stabilize v1v2 to v3 along withdestabilizing β21 from adopting CD4-bound-conformation 191 W, F Y191W,Y191F 4, 5 CL: SM: Stabilize V1V2 cap 198 F T198F 713 CL: V1V2-gp120core interface 201 F, Y, L, M, V I201W 173 CL: parallel β # SM: preventbridging sheet formation 202 F, W T202F, T202W 714 CL: V1V2-gp120 coreinterface, Substitute F or W, 203 F, W, Y, L, I, M, V Q203V 129 CL:Under V1V2 cap at N-term of β2 SM: stabilize prefusion cap/preventbridging sheet formation, β2 extends in CD4-bound state, this stabilizessmall hydrophobic pocket in ground state 204 F, W A204F, A204W 716, 724CL: V1V2-gp120 core interface, Substitute F or W, 208 W, Y, F, M V208W,93-96 CL: between α1 and strand leading to bridging sheet in the V208Y,V208F, CD4-bound conformation V208 SM: Destabilize CD4-boundconformation 209 R S209R, 196 CL: Loop between β3 and β4 SM: Fill cavityotherwise filled by CD4 induced α0 formation 220 F, Y, L, I, M, V, WP220W 180 CL: SM: stabilize gp41 interface 223 Y, W F223W  31, 780 CL:near β5 SM: stabilize gp120/gp41 interface 245 F V245F 700 CL: gp120 C2.Substitute F to stabilize gp120 V1/V2/V3, interprotomer 246 F, W, Y, L,I, M, V Q246W 197 CL: gp120-gp41 interface, near middle of β8 SM: Fill acavity between gp120 and gp41 to stabilize gp41 disordered region andgp120/gp41 interaction 254 F V254F 670 CL: gp120 C2 with F substitutionto stabilize gp120 260 F L260F 671 CL: gp120 C2 with F substitution tostabilize gp120 261 F L261F 672 CL: gp120 C2 with F substitution tostabilize gp120, interprotomer 263 W G263W 673 CL: gp120 C2 with Wsubstitution to stabilize gp120, interprotomer 302 F, W N302F, N302W725, 726 CL: V3-V1V2/gp120 core interface 309 F, W, Y, L, M I309W 136CL: Hydrophobic patch at C-term of V1V2 strand B SM: Stabilize V1V2strand B to V3 near trimeric interface 317 W, Y F317W 134 CL: Primaryhydrophobic pocket between V1V2 and V3 near position 159 SM: Stabilizehydrophobic core of V1V2-V3 interactions 323 W I323W 690 CL: gp120 V3.Substitute F to stabilize V1/V2/V3 326 M, W, F, Y, R I326R, I326F  45,691 CL: at the interface of V1V2 and V3 loops SM: Stabilize interactionsbetween V1V2 and V3 loops in the mature closed state 328 328: F, W, Y,L, I, Q328W 132 CL: near V3 loop M, V SM: Adding hydrophobic patch at V1loop to V3 loop 332 F, Y, W I332F 198 CL: C-term of beta V3B, SM: Fillscavity between V3 and gp120 core to stabilize closed V1V2 cap 380 FG380F 667 CL: gp120 C4 with F substitution to stabilize gp120 421 421:F, W, Y K421W, 123 CL: V1V2 interaction near residue 180 with V3 andbase of β21 SM: This will stabilize v1v2 to v3 along with destabilizingβ21 from adopting CD4-bound-conformation 423 F, Y, L, M, V, W I423F,I423W 173, 717, CL: gp120core-V1V2 interface, parallel β # 718 SM:prevent bridging sheet formation 426 G, V, I, L, F, Y, W, M426W, 54, 75,CL: CD4 binding site R, K, P M426A, 78, 83 SM: Constrains CD4 bindingloop, Blocks transition to CD4 M426F, bound conformation M426P 429 F, Y,L, I, M, V, W R429W 182 CL: under parallel β# SM: prevent bridging sheetformation 431 Any G431P  68, 782 CL: CD4 binding site SM: stericallyinterferes with CD4 binding specifically without affecting antibodybinding 432 F, W Q432F, Q432W 719, 720 CL: stabilize unligandedconformation of bridging sheet region 436 M, F, W A436M, A436F, 721,722, CL: stabilize unliganded conformation of bridging sheet regionA436W 723 437 F P437F 668 CL: gp120 C terminus with F substitution andstabilize gp120 C terminus 473 Any G473A, G473S, 65-67, CL: CD4 bindingsite G473Y 781 SM: sterically interferes with CD4 binding specificallywithout affecting antibody binding 478 F N478F 660 CL: gp120 C terminussubstituted with F and stabilize gp120 C terminus 522 Y F522Y  34 CL:near N-tern of fusion peptide SM: Fusion Peptide Cavity Fill 523 F L523F 33 CL: near N-tern of fusion peptide SM: Fusion Peptide Cavity Fill 530W M530W  29 CL: gp41-tryptophan clasp SM: stabilize interactions withingp41-tryptophan clasp 534 I, W, F, A, M, V S534V, S534A 47-48 CL:gp120/gp41 interface SM: fill cavity and add hydrophobic interactions atthe gp120- gp41 interactive surface 544 F, Y, W L544Y  32 CL: gp41 tipof α6 SM: stabilize gp120/gp41 interface

The recombinant HIV-1 Env ectodomain included in the trimer can includeany one of the cavity filling substitution at one of the HIV-1 Envpositions listed in Table 8. The recombinant HIV-1 Env ectodomain canalso include a combination of two or more (e.g., 3, 4, 5, 6, 7, or 8) ofthe cavity filling substitutions provided in Table 8 to stabilize theHIV-1 Env ectodomain trimer in the prefusion mature closed conformation.

Additional Substitutions

In some embodiments, the recombinant HIV-1 Env ectodomain can includeone or more amino acid substitutions that destabilize the C14-bound openconformation of the H6-Env ectodomain timer (and thereby preventtransition of the trimer from the prefusion closed conformation to theCD4-bound open conformation).

For example, in some embodiments the recombinant HIV-1 Env ectodomaincan include an amino acid substitution that introduces a proline residuein the β21 sheet (e.g., positions 430-435), such as a prolinesubstitution at position 432 or 433.

Exemplary HIV-1 Env positions for introducing an amino acidsubstitutions that destabilizes the CD4-bound open conformation of theHIV-1 Env ectodomain timer are provided in Table 7, as are correspondingamino acid substitutions with reference to the BG505 strain, and anexemplary sequence including the indicated substitution.

TABLE 7 Destabilization of CD4-induced- and stabilization of prefusionmature conformations Exem- Substi- Exem- plary tution plary substi-compared SEQ tution to BG505 ID NO 112I W112I Stabilize V1V2 116 112MW112M Stabilize V1V2 117 120T V120T Stabilize V1V2 149 122K L122KStabilize V1V2 150 120P V120P Stabilize V1V2 148 202P T202P StabilizeV1V2 147 427I W427I Stabilize prefusion closed conformation 118 427MW427M Stabilize prefusion closed conformation 119 429N R429N Stabilizeprefusion closed conformation 120 429L R429L Stabilize prefusion closedconformation 116 432P Q432P Destabilizes CD4-bound conformation 7, 775432E Q432E Destabilizes CD4-bound conformation 42 432D Q432DDestabilizes CD4-bound conformation 43 433P A433P Destabilizes CD4-boundconformation 6, 774 434P 434P Destabilizes CD4-bound conformation 44435P 435P Destabilizes CD4-bound conformation 45 436P 436P DestabilizesCD4-bound conformation 46 437A P437A Destabilizes CD4-bound conformation47 438A P438A Destabilizes CD4-bound conformation 48 474A D474AStabilize prefusion closed conformation 118 476A R476A Stabilizeprefusion closed conformation 124

In several embodiments, the recombinant HIV-1 Env ectodomain can includeone or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of the amino acidsubstitutions as listed in Table 7 to destabilize the CD4-inducedconformation of the HIV-1 ectodomain trimer (and thereby stabilize theHIV-1 Env ectodomain trimer in the prefusion mature closedconformation). Additionally, any of the substitutions shown in Table 7can be combined with the other stabilizing substitutions describedherein to stabilize the HIV-1 Env ectodomain trimer in the prefusionmature closed conformation.

In some embodiments, the recombinant HIV-1 Env ectodomain includes oneor more amino acid substitutions that destabilize the formation of theα0 helix. As described in Example 1, the α0 helix (˜ residues 64-74) ispresent in the CD4-bound open conformation, but not the prefusion matureclosed conformation, of trimeric HIV-1 Env. In some embodiments, therecombinant HIV-1 Env ectodomain includes a proline amino acidsubstitution at position 66 or 67, or both positions 66 and 67 (such asa H66P and/or N67P substitution) that disrupts formation of the α0 helixin the recombinant HIV-1 Env ectodomain trimer. Exemplary sequencesincluding these mutations are set forth as SEQ ID NOs: 102-104.

In some embodiments, the recombinant HIV-1 Env ectodomain includes anon-natural disulfide bond between a pair of cysteine substitutions atone of positions 57-58 and position 77, or between position 66 or 68 andone of positions 207-209, wherein the non-natural disulfide bonddestabilizes or disrupts formation of the α0 helix in the recombinantHIV-1 ectodomain. In some embodiments, the recombinant HIV-1 Envectodomain can include a non-natural disulfide bond between a pair ofcysteine substitutions at one or more of the following sets positions:55 and 77, 57 and 77, 58 and 77, 66 and 207, 66 and 208, 66 and 209, 68and 209, and 68 and 208, wherein the non-natural disulfide bond disruptsformation of the α0 helix in the recombinant HIV-1 ectodomain. In someembodiments, the recombinant HIV-1 Env ectodomain can include one ormore of the following sets of amino acid substitutions: A55C and T77C,D57C and T77C, A58C and T77C, V66C and K207C, V66C and S209C, V68C andS209C, and V68C and V208C. Exemplary sequences including these mutationsare set forth as SEQ ID NOs: 87, 98-101, and 683.

In more embodiments, the recombinant HIV-1 Env ectodomain can include aproline amino acid substitution at position 66 or 67, or both positions66 and 67 (such as a H66P and/or N66P substitutions), and furtherinclude a pair of cysteine substitutions at positions 57 and 77, 58 and77, 68 and 208, or 68 and 209, the combination of which disruptsformation of the α0 helix in the recombinant HIV-1 ectodomain. Forexample, in some embodiments, the recombinant HIV-1 Env ectodomain caninclude one of the following sets of amino acid substitutions:D57C/T77C, H66P, N67P; A58C/T77C, H66P, N67P; V68C/S209C, H66P, N67P;V68C/V208C, H66P, N67P; or V68C/S209C, H66P, N67P. Exemplary HIV-1 Envectodomain sequences including these mutations are set forth as SEQ IDNOs: 105-108 and 109.

Stabilizing Gp41

In additional embodiments, the recombinant HIV-1 Env ectodomain caninclude one or more disulfide bonds that stabilize gp41 in the matureclosed prefusion conformation. Exemplary mutations include those thatcan form a non-natural gp120-gp41 disulfide bond or a non-naturalgp41-gp41 disulfide bond. Exemplary HIV-1 Env positions that can bemutated to cysteine to form such stabilizing disulfide bonds, as well asexemplary mutations (in the context of the BG505 strain), and sequencesincluding the indicated mutations are provided in Table 9, below.

TABLE 9 Non-natural gp120-gp41 and gp41 disulfide bonds Intra- orExemplary Env Exemplary Inter- Exemplary positions Substitutionsprotomer? Comment SEQ ID NO gp120-gp41 disulfide bonds 41 and 540G41C/Q540C Intra Stabilize α6/Prevent HR1 formation 14 41 and 541G41C/A541C Intra Stabilize α6/Prevent HR1 formation 199, 200, 201 43 and526 P43C/A526C Intra Stabilize α6/Prevent HR1 formation 15 51 and 574T51C/K574C Intra Stabilize α7/Prevent HR1 formation 151 53 and 574F53C/K574C Intra Stabilize α7/Prevent HR1 formation 152 51 and 578T51C/A578C Intra Stabilize α7/Prevent HR1 formation 153  43and 540P43C/Q540C Intra Stabilize α6/Prevent HR1 formation 18 88 and 527N88C/G527C Intra Stabilize fusion peptide 17 107 and 574  D107C/K574CIntra Stabilize α7/Prevent HR1 formation 166 220 and 578  P220C/A578CIntra Stabilize α7/Prevent HR1 formation 10 221 and 582  A221C/A582CIntra Stabilize α7/Prevent HR1 formation 11, 16 428 and 560  Q428C/E560CInter Stabilize α6/α7 linker/Prevent HR1 163 formation 428 and 561 Q428C/A561C Inter Stabilize α6/α7 linker/Prevent HR1 164 formation 428and 562  Q428C/Q562C Inter Stabilize α6/α7 linker/Prevent HR1 165formation 498 and 610  498C/W610C Intra Prevent HR1/2 formation 13 36and 606 V36C/T606C Intra Prevent HR1 formation 647 37 and 606 T37C/T606CIntra] Prevent HR1 formation 648 41 and 537 G41C/L537C Intra Prevent HR1formation 734 41 and 541 G41C/A541C Intra Prevent HR1 formation 736 43and 526 P43C/A526C Intra Prevent HR1 formation 737 73 and 572 A73C/G572CIntra Prevent HR1 formation 738 84 and 521 I84C/G521C Intra Prevent HR1formation 739 89 and 527 V89C/G527C Intra Prevent HR1 formation 740 73and 572 A73C/G572C Intra Prevent HR1 formation 741 84 and 521 I84C/G521CIntra Prevent HR1 formation 742 89 and 527 V89C/G527C Intra Prevent HR1formation 743 gp41 disulfide bonds 550 and 575  Q550C/Q579C IntraPrevent HR1 formation 167, 169 551 and 575  Q551C/Q575C Intra PreventHR1 formation 168

Any one or more of the pairs of cysteine substitutions listed in Table 9can be included on a recombinant HIV-1 Env ectodomain to generate arecombinant HIV-1 Env ectodomain trimer stabilized in the prefusionmature closed conformation. Further, the recombinant HIV-1 Envectodomain can include any one or more of the pairs of cysteinesubstitutions listed in Table 9 in combination with any of the otherstabilizing mutations disclosed herein to generate a recombinant HIV-1Env ectodomain that can form a trimer stabilized in the prefusion matureclosed conformation.

Additionally, the recombinant HIV-1 Env ectodomain can include the SOS(501C and 605C), IP (559P), and/or SOSIP (501C, 605C, 559P)substitutions in combination with any of the stabilizing mutationsdisclosed herein to generate a recombinant HIV-1 Env ectodomain that canform a trimer stabilized in the prefusion mature closed conformation.

Exemplary Combinations

In several embodiments, any two or more of the HIV-1 Env mutationsdisclosed herein can combined to generate the recombinant HIV-1 Envectodomain that can form a trimer stabilized in the prefusion matureclosed conformation.

For example, in some embodiments, the recombinant HIV-1 Env ectodomaincan include a non-natural disulfide bond that stabilizes the protein ina PGT122-bound conformation (e.g., with a V1V2 domain in a mature closedconformation; such as a non-natural disulfide bond between one or moreof positions 201 and 433) and further include one or more amino acidsubstitutions that stabilize gp41 in a mature closed conformation (e.g.,with distinct α6 and α7 helices; such as substitutions listed in Table9, such as 41C/540C substitutions or a SOS, IP, SOSIP substitution). Infurther embodiments, the recombinant HIV-1 Env ectodomain can include anon-natural disulfide bond that stabilizes the protein in a PGT122-boundconformation (e.g., with a V1V2 domain in a mature closed conformation;such as a non-natural disulfide bond between one or more of positions201 and 433) and includes one or more mutations that destabilize the CD4binding domain, such as a substitution at position 473, and/or includesa cavity filling substitution, and further includes one or more aminoacid substitutions that stabilize gp41 in a mature closed conformation(e.g., with distinct α6 and α7 helices; such as substitutions listed inTable 9, such as 41C/540C substitutions or a SOS, IP, SOSIPsubstitution).

In some embodiments, the recombinant HIV-1 Env ectodomain includes apair of cysteine substitutions at one of positions 198-202 and one ofpositions 428-437 that form a non-natural disulfide bond, and furtherincludes one or more amino acid substitutions that stabilize gp41 asdescribed above (e.g., as listed in Table 9, such as 41C/540Csubstitutions), and/or can further include the SOS, IP, SOSIP mutations.In a non-limiting embodiment, the recombinant HIV-1 Env ectodomainincludes 201C, 433C, 501C, 605C, and 559P substitutions (such as I201C,A433C, A501C, T605C, and I559P substitutions).

In some embodiments, the recombinant HIV-1 Env ectodomain includes apair of cysteine substitutions at one of positions 174 and 319, 195 and433, 199 and 433, 199 and 431, 201 and 433, 221 and 582, or 304 and 440,that form a non-natural disulfide bond, and further includes one or moreamino acid substitutions that stabilize gp41 as described above (e.g.,as listed in Table 9, such as 41C/540C substitutions), and/or canfurther include the SOS, IP, SOSIP mutations. In a non-limitingembodiment, the recombinant HIV-1 Env ectodomain includes 201C, 433C,501C, 605C, and 559P substitutions (such as I201C, A433C, A501C, T605C,and I559P substitutions). In some such embodiments, the recombinantHIV-1 Env ectodomain can further includes a tryptophan substitution atposition 223, or a proline substitution at position 432 or 433.

In more embodiments, the recombinant HIV-1 Env ectodomain includes twopairs of cysteine substitutions at one of positions (i) 195 and 433, and304 and 440; (ii) 195 and 433, and 174 and 319; (iii) 199 and 433, and304 and 440; (iv) 199 and 433, and 174 and 319; (v) 201 and 433, and 304and 440; (vi) 201 and 433, and 174 and 319; that form two non-naturaldisulfide bonds, and can further includes one or more amino acidsubstitutions that stabilize gp41 as described above (e.g., as listed inTable 9, such as 41C/540C substitutions), and/or can further include theSOS, IP, SOSIP mutations. In a non-limiting embodiment, the recombinantHIV-1 Env ectodomain includes 201C, 433C, 501C, 605C, and 559Psubstitutions (such as I201C, A433C, A501C, T605C, and I559Psubstitutions). In some such embodiments, the recombinant HIV-1 Envectodomain can further includes a tryptophan substitution at position223, or a proline substitution at position 432 or 433. Exemplaryimmunogens include those with a BG505gp140.6R.SOSIP.664 backgroundsequence further including 1201C/A433C and R304C/Q440C substitutions;S199C/A433C and R304C/Q440C substitutions; I201C/A433C and V120C/Q315Csubstitutions; G473P and V120C/Q315C substitutions; G473Y andV120C/Q315C substitutions; G473P and R304C/Q440C substitutions; G473Yand R304C/Q440C substitutions; N425C/A433C and V120C/Q315Csubstitutions; and I201C/A433C and V120C/Q315C substitutions.

In additional embodiments, the recombinant HIV-1 Env ectodomain caninclude a combination of two or more non-natural disulfide bonds betweenpairs of cysteine substitutions as described above that (e.g., as listedin Table 6). Non-limiting examples of combinations of two or morenon-natural disulfide bonds between pairs of cysteine substitutions areprovided in Table 10. In some embodiments, the recombinant HIV-1 Envectodomain can include one or more pairs of cysteine substitutions asdescribed above that form a non-natural disulfide bond (e.g., as listedin Table 6), or a combination of pairs of cysteine substitutions aslisted in Table 10, and further includes one or more amino acidsubstitutions that stabilize gp41 as described above (e.g., as listed inTable 9, such as 41C/540C substitutions), and/or can further include theSOS, IP, SOSIP mutations.

TABLE 10 Exemplary combinations of cysteine substitutions to generatenon-natural disulfide bonds Exemplary Positions Exemplary substitutionsSEQ ID NO 200C/313C, 51C/574C A200C/P313C, T51C/K574C 151 200C/313C,53C/574C A200C/P313C, F53C/K574C 152 200C/313C, 51C/578C A200C/P313C,T51C/A578C 153 128C/165C, 200C/313C, T128C/L165C, A200C/P313C, 15451C/574C T51C/K574C 128C/165C, 200C/313C, T128C/L165C, A200C/P313C, 15553C/574C F53C/K574C 128C/165C, 200C/313C, T128C/L165C, A200C/P313C, 15651C/578C T51C/A578C

In some embodiments, the recombinant HIV-1 Env ectodomain can include anon-natural disulfide bond that stabilizes the protein in a PGT122-boundconformation (e.g., with a V1V2 domain in a mature closed conformation;such as a non-natural disulfide bond between one or more of positions128 and 167, 174 and 318, 175 and 319, 204 and 434, 204 and 436, or 318and 437) and further includes one or more mutations that destabilize theCD4 binding domain, such as a substitution at position 473 (such as aG473A substitution). For example, in some embodiments, the recombinantHIV-1 Env ectodomain can include one of the following sets of amino acidsubstitutions: T128C/T167C and G473A, S174C/A318C and G473A, L175C/A319Cand G473A, A204C/M434C and G473A, A204C/A436C and G473A, or Y318C/P437Cand G473A. In additional such embodiments, the recombinant HIV-1 Envectodomain can further include one or more amino acid substitutions thatstabilize gp41 as described above (e.g., as listed in Table 9, such as41C/540C substitutions), and/or can further include the SOS, IP, SOSIPmutations. Exemplary gp140 sequences including such substitutions areset forth as SEQ ID NOs: 53, 55, 56, 58, 59, 72.

In additional embodiments, the recombinant HIV-1 Env ectodomain caninclude a cavity filling substitution as described above, and furtherincludes an amino acid substitution that destabilizes the CD4-inducedconformation of HIV-1 Env, such as a A433P substitution. In additionalsuch embodiments, the recombinant HIV-1 Env ectodomain can furtherinclude one or more amino acid substitutions that stabilize gp41 asdescribed above (e.g., as listed in Table 9, such as 41C/540Csubstitutions), and/or can further include the SOS, IP, SOSIP mutations.In a non-limiting embodiment, the recombinant HIV-1 Env ectodomainincludes 433P, 501C, 605C, and 559P substitutions (such as A433P, A501C,T605C, and I559P substitutions).

In additional embodiments, the recombinant HIV-1 Env ectodomain includesa combination of two or more of the stabilizing substitutions describedherein. For example, the recombinant HIV-1 Env ectodomain can include429L and 427M (e.g., R429L and W427M substitutions), 437A and 438A(e.g., P437A and P438A substitutions), or 474A and 476A (e.g., D474A andR476A substitutions). In additional such embodiments, the recombinantHIV-1 Env ectodomain can further include one or more amino acidsubstitutions that stabilize gp41 as described above (e.g., as listed inTable 9, such as 41C/540C substitutions), and/or can further include theSOS, IP, SOSIP mutations. Exemplary sequences including thesesubstitutions include those provided as SEQ ID NOs: 49, 115, and 122.Additionally, any of the above cavity filling substitutions can becombined with the other stabilizing substitutions described herein tostabilize the HIV-1 Env ectodomain trimer in the prefusion mature closedconformation. For example, in some embodiments, the recombinant HIV-1Env ectodomain includes a cavity filling substitution as describedabove, such as at one or more of gp120 positions 50, 110, 114, 117, 117,120, 121, 121, 123, 159, 220, 426, 220, 429, and further includessubstitutions to introduce a non-natural disulfide bond, such asI201C/A433C substitutions. In some non-limiting embodiments, therecombinant HIV-1 Env ectodomain includes a non-natural disulfide bondbetween I201C and A433C substitutions and further includes one of aT50W, S110W, Q114W, K117W, K117E, V120W, K121W, K121E, T123W, F159Y,M426W, P220W, or R429W cavity filling amino acid substitution. In someembodiments, the recombinant HIV-1 Env ectodomain includes a cavityfilling substitution at position 193 (e.g., L193F), and further includessubstitutions to introduce a non-natural disulfide bond betweenpositions 195 and 423, such as N195C/I423C substitutions; an exemplarysequence is set forth as SEQ ID NO: 688. In some embodiments, therecombinant HIV-1 Env ectodomain includes a cavity filling substitutionat position 431 (e.g., G431F), and further includes substitutions tointroduce a non-natural disulfide bond between positions 202 and 434,such as T202C/M434C substitutions; an exemplary sequence is set forth asSEQ ID NO: 655. In some non-limiting embodiments, the recombinant HIV-1Env ectodomain includes a non-natural disulfide bond between I201C andA433C substitutions and further includes one of a T50W, S110W, Q114W,K117W, K117E, V120W, K121W, K121E, T123W, F159Y, M426W, P220W, or R429Wcavity filling amino acid substitution. In additional such embodiments,the recombinant HIV-1 Env ectodomain can further include one or moreamino acid substitutions that stabilize gp41 as described above (e.g.,as listed in Table 9, such as 41C/540C substitutions), and/or canfurther include the SOS, IP, SOSIP mutations. Exemplary recombinantHIV-1 Env ectodomain sequences including such substitutions are setforth as SEQ ID NOs: 120, 183-194.

In several embodiments, the recombinant HIV-1 Env ectodomain can includea combination of substitutions as set forth in Table 11, that include atleast one cavity filling substitution.

TABLE 11 Exemplarity cavity-filling amino acid substitutions ExemplaryExemplary Exemplary Position substitutions Substitutions SEQ ID NOCavity Location (CL) and stabilizing mechanism (SM) 120, 203 201: V120W,Q203V 129 CL: Under V1V2 cap at N-term of β2 F, W, Y, L, I, M; SM:stabilize prefusion cap/prevent bridging sheet formation, 203: β2extends in CD4-bound state, this stabilizes small F, W, Y, L, I, M, Vhydrophobic pocket in ground state  39, 534 39: W, M, I, F Y39F, S534V;47-48 CL: gp120/gp41 interface 534: I, W, F, A, M, Y39W, S534A SM: fillcavity and add hydrophobic interactions at the gp120- V gp41 interactivesurface 39, 534 + 39: W, M, I, F; Y39F, S534V, 49 CL: gp120/gp41interface T37V, 534: I, W, F, A, M, T37V, T499V SM: fill cavity and addhydrophobic interactions at the gp120- T499V V gp41 interactive surface39, 534 + 39: W, M, I, F; Y39F, Y40F, 50 CL: gp120/gp41 interface Y40F,534: I, W, F, A, M, S534V, T37V, SM: fill cavity and add hydrophobicinteractions at the gp120- T37V, V T499V gp41 interactive surface T499V 53, 246 246: F, W, Y, L, I, F53W, Q246W 197 CL: gp120-gp41 interface,N-term of β2, middle of β8 M, V SM: Fill a cavity between gp120 and gp41to stabilize gp41 disordered region and gp120/gp41 interaction   68,209, 68: F, W, Y, L, I, M V68L, S209R 196 CL: Loop between α1 and β0 andloop between β3 and β4 SM: Fill cavities that are otherwise filled byCD4 induced α0 formation 125, F, W, Y, L, I, M, V L125W_deltaP124 131CL: Cavity between N-term of V1V2 domain and V3 near delP124 residue 127and 126-196 disulfide of V1V2 SM: Removal of ground statedestabilization/flexibility cavity filling/proline removal 139, 326 139:F. M, I, Y, T139W, I326R 45 CL: at the interface of V1V2 and V3 loops T;SM: Stabilize interactions between V1V2 and V3 loops in the 326: M, W,F, mature closed state Y, R 151, 153, 153: F, W, Y, L, I, R151E, E153W,132 CL: V1 loop at position 153 328 M, V; Q328W SM: Adding hydrophobicpatch at V1 loop to V3 loop 328: F, W, Y, L, I, M, V 177, 323 323: F, Y,W Y177W, I332F 198 CL: C-term of beta C, C-term of beta V3B, SM: Fillscavity between V3 and gp120 core to stabilize closed V1V2 cap 180, 421,421: F, W, Y; 180: D180L, K421W 123 CL: V1V2 interaction near residue180 with V3 and base of L, V, I, M β21 SM: This will stabilize v1v2 tov3 along with destabilizing β21 from adopting CD4-bound-conformation201, 423 F, Y, L, M, V I201W, I423W 173 CL: parallel β # SM: preventbridging sheet formation 223, 544 223: Y, W F223W, L544Y 32 CL: gp41 tipof α6 544: F, Y, W SM: stabilize gp120/gp41 interface L125, 195  WL125W, I195W 701 CL: V1V2-V3 interface near 126-196 disulfide bond 176,323 176: Y, W; 323: F176W/I323Y 730 CL: gp120 V1/V2/V3, stabilizeV1/V2/V3 F, Y, W 176, 154 176: Y, W; 154: F176W/L154W 731 CL: gp120V1/V2, stabilize V1/V2/V3 F, Y, W 159, 154 159: Y, W; 154: F159Y/L154W732 CL: gp120 V1/V2, stabilize V1/V2/V3 F, Y, W I251, 260  F I251F/L260F658 CL: Engineering hydrophobic core between gp120 V2/V3 with double Fsubstitution to stabilize V1/V2/V3

In some embodiments, the recombinant HIV-1 Env ectodomain comprisesgp120-gp41 protomers comprising the SOSIP and 201C/433C substitutionsand further comprising a cavity filling substitution (such as a Y F, orM substitution) at anyone of positions: 70; 75; 110; 111; 112; 115; 117;118; 120; 153; 154; 159; 164; 172; 175; 176; 179; 191; 193; 194; 198;202; 204; 208; 223; 304; 307; 309; 315; 316; 323; 423; 427; 430; 432;432; 436; 544; 580; 583; 159 and 323; 44 and 537; 544 and 223; 544, 537,and 223; 580 and 583; 125 and 194; 134, 175, 322, and 326; 134, 322, and326; 134, 136, 150, and 326; 154, 300, 302, and 320; 120, 203, and 318;120 and 315; 177 and 420; 177, 328, and 420; 116, 426, and 432; 426 and432; 134, 175, 322, 326, 136, and 150; 120, 203, 318, and 315; 154, 300,302, 320, 177, and 420; or 139, 140, 324, and 325.

In some embodiments, the recombinant HIV-1 Env ectodomain comprisesgp120-gp41 protomers comprising the SOSIP and 201C/433C substitutionsand further comprising a substitution to destabilize the CD34-inducedconformation, such as a F210A; F210S; Q432P; R429N; R429L; R429L andW427M; T202P; or V120T substitution

In some embodiments, the recombinant HIV-1 Env ectodomain comprisesgp120-gp41 protomers comprising the SOSIP and 201C/433C substitutionsand further comprising cysteine substitutions to introduce a non-naturaldisulfide bond, such as T538C and Q652C; R304C and Q440C; G431GC andS199C; A58C and T77C; D57C and T77C

In some embodiments, the recombinant HIV-1 Env ectodomain comprisesgp120-gp41 protomers Comprising the SOSIP and 201C/433C substitutionsand further comprising substitutions to disrupt formation of helix 0,such as W69P; V68P; T71P; N67P; H66P; or N67P and H66P In someembodiments, the recombinant HIV-1 Env ectodomain comprises gp120-gp41protomers comprising the SOSIP and 201C/433C substitutions and furthercomprising substitutions to destabilize the gp41 helix bundle, such asI573T; G594N; I573T and G594N; I573T and G594N and K574E; I573T, G594N,and K574T.

N-Linked Glycosylation Sites

In several embodiments, the recombinant HIV-1 Env ectodomain trimer canincludes one or more N-linked glycosylation sites introduced onto themembrane proximal portion of the trimer to mask non-neutralizingepitopes present on this portion of the trimer. Such mutations aretypically utilized in soluble embodiments of the recombinant HIV-1 Envectodomain trimer. To create an N-linked glycosylation site, thesequence Asn-X-Ser/Thr (where X is any amino acid except Pro) can to beintroduced. This can be accomplished by substitution of a Ser/Thr aminoacid two residues C-terminal to a native Asn residue, or by substitutionof an Asn amino acid two residues N-terminal to a native Ser/Thrresidue, or by substitution of both an Asn and Ser/Thr residue separatedby one non-proline amino acid. In some embodiments, the recombinantHIV-1 Env ectodomain comprises one or more amino acid substitutions thatintroduce an N-linked glycosylation site at the N-terminus of the gp120polypeptide, the C-terminus of the gp120 polypeptide, and/or theC-terminus of the gp41 polypeptide. Exemplary amino acid substitutionsfor introducing one or more such N-linked glycosylation sites areprovided under code D of the Table in Table 13, and are provided inTable 12, below:

TABLE 12 Exemplary N-linked glycan mutants to mask non-neutralizingepitopes. Glycan Exemplary Exemplary position substitutions SEQ ID NO504 504N/506T 453 661 661N/663T 454 504 and 661 504N/506T, 661N/663T 455502 K502N/R504T 456 658 Q658N/L660T 457  33 W35T 458  35 W35N 459  35and 504 W35N, R504N/V506T 460  33 and 661 W35T, L661N/L663T 461 502 and661 K502N/R504T, L661N/L663T 462

Antibody Stabilization

In additional embodiments, the disclosed immunogens can include arecombinant HIV-1 Env ectodomain trimer covalently linked (e.g., by anon-natural disulfide bond) to one or more neutralizing antibodies, suchas the VRC01, PGT122, or 35O22 antibodies. Linkage to the neutralizingantibody can increase the stability of the immunogen in the prefusionmature closed conformation.

35O22

In some embodiments, the recombinant HIV-1 Env ectodomain trimerincludes a recombinant HIV-1 Env ectodomain including a cysteinesubstitution that can form a non-natural disulfide bond with a cysteineresidue in the heavy or light chain variable region of the 35O22monoclonal antibody. For example, in some embodiments, the HIV-1ectodomain includes a cysteine substitution at position 90, which canform a non-natural disulfide bond with a cysteine at position 80 (kabatnumbering) of the 35O22 heavy chain variable region. In someembodiments, the HIV-1 ectodomain includes a cysteine substitution atposition 238, which can form a non-natural disulfide bond with acysteine at position 77 (kabat numbering) of the 35O22 heavy chainvariable region. In some embodiments, the HIV-1 ectodomain includes acysteine substitution at position 529, which can form a non-naturaldisulfide bond with a cysteine at position 111 (kabat numbering) of the35O22 heavy chain variable region. In some embodiments, the HIV-1ectodomain includes a cysteine substitution at position 624, which canform a non-natural disulfide bond with a cysteine at position 109 or 112(kabat numbering) of the 35O22 heavy chain variable region. In someembodiments, the HIV-1 ectodomain includes a cysteine substitution atposition 625, which can form a non-natural disulfide bond with acysteine at position 109 (kabat numbering) of the 35O22 heavy chainvariable region. Exemplary sequences for use in such embodiments areprovided under code C of the Table in Table 13. For example, exemplaryrecombinant HIV-1 Env ectodomain sequences including such cysteinesubstitutions are set forth as SEQ ID NOs: 401-405, and 411.Additionally, exemplary 35O22 heavy chain variable region sequences foruse in such embodiments are set forth as SEQ ID NOs: 406-410.

VRC01

In some embodiments, the recombinant HIV-1 Env ectodomain trimerincludes a recombinant HIV-1 Env ectodomain including a cysteinesubstitution that can form a non-natural disulfide bond with a cysteineresidue in the heavy or light chain variable region of the VRC01monoclonal antibody. In some embodiments, the recombinant HIV-1 Envectodomain trimer includes a recombinant HIV-1 Env ectodomain includinga cysteine substitution at position 449 (e.g., a G459C substitution,HXB2 numbering) that can form a non-natural disulfide bond with a heavychain variable region of a VRC01 monoclonal antibody comprising acysteine substitution at heavy chain position 60 or 61 (kabatnumbering). Exemplary sequences for use in such embodiments are providedunder code C of the Table in Table 13. For example, exemplaryrecombinant HIV-1 Env ectodomain sequences including a position 459cysteine substitution are set forth as SEQ ID NOs: 412-436. ExemplaryVRC01 heavy and light chain sequences for use in such embodiments (e.g.,including heavy chain variable region with a cysteine substitution atposition 60 or 61) are set forth as SEQ ID NOs: 437-448.

PGT122

In some embodiments, the recombinant HIV-1 Env ectodomain trimerincludes a recombinant HIV-1 Env ectodomain including a cysteinesubstitution that can form a non-natural disulfide bond with a cysteineresidue in the heavy or light chain variable region of the PGT122monoclonal antibody. In some embodiments, the recombinant HIV-1 Envectodomain trimer includes a recombinant HIV-1 Env ectodomain includinga cysteine substitution at position 323 (e.g., a I323C substitution,HXB2 numbering) that can form a non-natural disulfide bond with a heavychain variable region of a PGT122 monoclonal antibody comprising acysteine substitution at heavy chain position 29 or 67 (kabatnumbering). Exemplary sequences for use in such embodiments are providedunder code C of the Table in Table 13. For example, exemplaryrecombinant HIV-1 Env ectodomain sequences including a position 323cysteine substitution are set forth as SEQ ID NOs: 449-450. ExemplaryVRC01 heavy and light chain sequences for use in such embodiments (e.g.,including heavy chain variable region with a cysteine substitution atposition 29 or 67) are set forth as SEQ ID NOs: 451-452.

Chimeric Env Ectodomains

In some embodiments, the recombinant HIV-1 Env ectodomain stabilized inthe prefusion mature closed conformation can include sequences frommultiple strains of HIV-1. For example, the recombinant HIV-1 Envectodomain can include a gp120 sequence from a first HIV-1 strain and agp41 sequence from a heterologous HIV-1 strain, or a particularstructural domain (such as the V1V2 domain) from a HIV-1 strain ofinterest (such as CAP256.SU, a BB201.B42, a KER2018.11, a CH070.1, aZM233.6, a Q23.17, a A244, a T250-4, or a WITO.33) with the remainder ofthe HIV-1 Env ectodomain from a heterologous HIV-1 strain (such asBG505). The chimeric HIV-1 Env ectodomain can further include any of theamino acid substitutions described herein, for example the 201C/433C,SOSIP, and DS substitutions for stabilization in the prefusion matureclosed conformation. In the context of inducing an immune response in asubject that can control infection across multiple HIV-1 strains, theuse of immunogens based on diverse HIV-1 strains can overcome theintrinsic sequence diversity of HIV-1 Env. Exemplary sequences ofrecombinant HIV-1 Env ectodomains linked to a nanoparticle subunit areprovided under code H in Table 13 included in Example 15.

Exemplary sequences of chimeric HIV-1 Env ectodomain trimers thatinclude the V1V2 domain sequence (positions 126-196) of the CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, orWITO.33 strains of HIV-1, with the remainder includingBG505.SOSIP.DS.368R sequence, are provided as follows: Q23.17 V1V2chimera (SEQ ID NO: 2126), ZM233.6 V1V2 chimera (SEQ ID NO: 2125),WITO.33 V1V2 chimera (SEQ ID NO: 2128), A244 V1V2 chimera (SEQ ID NO:2127), BB201.B42 V1V2 chimera (SEQ ID NO: 2122), KER2018.11 V1V2 chimera(SEQ ID NO: 2123), CH070.1 V1V2 chimera (SEQ ID NO: 2124), CAP256.SUV1V2 chimera (SEQ ID NO: 2121), and T-250-4 V1V2 chimera (SEQ ID NO:2129).

Platform

As described in the Examples, prefusion mature gp41 wraps itshydrophobic core around extended N- and C-termini-strands of gp120 (seeFIGS. 11 and 38 ). Accordingly, in some embodiments, the recombinantHIV-1 Env ectodomain trimer can include a membrane proximal “platform”including the N- and C-terminal regions of gp120, and the gp41ectodomain, from a first HIV-1 strain (such as BG505), and the remainderof gp120 from one or more heterologous HIV-1 strains. This chimericdesign allows for production of heterogeneous HIV-1 Env proteins thatcomprise membrane distal features of interest (such as the V1V2 domain,V3 domain, and CD4 binding site).

In some embodiments, the recombinant Env ectodomain includes N- andC-terminal regions of gp120 as well as the gp41 ectodomain from a firstHIV-1 strain (such as BG505, for example, with SOSIP substitutions), andthe remainder of gp120 from a heterologous HIV-1 strain. In someembodiments, the heterologous HIV-1 strain can be a subtype A (such asB1369.9A, MB201.A1, QH209.14M.A2), subtype B (such as AC10.29), subtypeC (such as 0921.V2.C14, 16055-2.3, 25925-2.22, 286.36, CAP45.G3, CNE58,DU156.12, DU422.01, MW965.26, ZM53.12, ZM55.28a, ZM106.9), subtype CRFAC (such as 3301.V1.C24, 6545.V4.C1), subtype CFR AE (such as 620345.c1,C1080.c3, C4118.09, CNE55, TH966.8) and subtype CRF BC (such asCH038.12, CH117.4) strain of HIV-1.

In some embodiments, the recombinant HIV-1 Env ectodomain can include agp41 ectodomain, an N-terminal region of the gp120 polypeptidecomprising a β-4 strand and a C-terminal region of the gp120 polypeptidecomprising a β26 strand from a first strain of HIV-1 (such as BG505),and all or a portion of the remaining residues of the gp120 polypeptideare from one or more heterologous HIV-1 strains. The heterologous straincan be, for example, one of CAP256.SU (SEQ ID NO: 51), a BB201.B42 (SEQID NO: 81), a KER2018.11 (SEQ ID NO: 107), a CH070.1 (SEQ ID NO: 174), aZM233.6 (SEQ ID NO: 745), a Q23.17 (SEQ ID NO: 746), a A244 (SEQ ID NO:747), a T250-4 (SEQ ID NO: 2114), a WITO.33 (SEQ ID NO: 748), a 426c(with N276D, N460D, N463D, SEQ ID NO: 2144, a d45-01dG5 (2145), or aJRFL (SEQ ID NO: 2115) strain of HIV-1. In additional embodiments, theN-terminal region of the gp120 polypeptide can further include the β-3strand from the first HIV-1 strain (such as BG505). In more embodimentsthe C-terminal region of the gp120 polypeptide can further include theβ25 strand or the β25 strand and all or a portion of the α5 helix fromthe first HIV-1 strain (such as BG505). In more embodiments, theN-terminal region of the gp120 polypeptide can include from 5 to 30(such as 10, 30, 5-20, 5-25, 5-15, 5-10, 10-20, 20-30, 15-25, or 5, 10,15, 20, 25) amino acids and/or the C-terminal region of the gp120polypeptide can include from 5-40 (such as 10-40, 5-30, 5-25, 5-20,10-20, 20-30, 30-40, 10-30, 20-40, or 5, 10, 15, 20, 25, 30, or 35)amino acids, from the N- or C-terminus of the gp120 polypeptide,respectively, from the first strain of HIV-1 (such as BG505). Any of thestabilizing amino acid substitutions (such as the SOSIP substitutions,and/or the 201C/433C substitutions) can be included in the chimericHIV-1 Env ectodomain.

In some embodiments, the recombinant Env ectodomain can include gp120residues 31-45 and 478-507, and gp41 residues (e.g., 512-664) from thefirst HIV-1 strain (such as BG505), and the remainder of the gp120residues in the Env protein can be from a heterologous HIV-1 strain. Forexample, the recombinant Env ectodomain can include gp120 positions31-45 and 478-507, and gp41 residues (e.g., 512-664) from the BG505strain with SOSIP substitution (e.g., as set forth as SEQ ID NO: 3), andthe remaining gp120 residues in the Env ectodomain can be from any oneof the CAP256.SU, BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244,WITO.33, JRFL, 426c (with N276D, N460D, N463D), d45-01dG5, B1369.9A,MB201.A1, QH209.14M.A2, 0921.V2.C14, 16055-2.3, 25925-2.22, 286.36,CAP45.G3, DU156.12, DU422.01, MW965.26, ZM53.12, ZM55.28a, ZM106.9,3301.V1.C24, 6545.V4.C1, 620345.c1, C1080.c3, C4118.09, CNE55, TH966.8,AC10.29, CH038.12, CNE58, or CH117.4 strains of HIV-1. Any of thestabilizing amino acid substitutions (such as the SOSIP substitutions,and/or the 201C/433C substitutions) can be included in the chimericHIV-1 Env ectodomain. Non-limiting examples of sequences of suchchimeric HIV-1 Env ectodomains (that may also include one or more aminoacid substitutions, such as 201C/433C and SOSIP substitutions tostabilize the HIV-1 ectodomain in the prefusion mature closedconformation) are provided herein as SEQ ID NOs: 379-386, 387, 764-772,and 856-1036. Thus, in some embodiments, the recombinant HIV-1 Envprotein includes an amino acid sequence set forth as any one of SEQ IDNOs: 379-386, 387, 764-772, and 856-1036, or an amino acid sequence atleast 80% (such as at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs:379-386, 387, 764-772, and 856-1036. In a preferred embodiment, therecombinant HIV-1 Env protein includes an amino acid sequence set forthas any one of SEQ ID NOs: 856, 872, 881, 888, 902, 908, 917, 924, 930,933, 937, 938, 940, 953, 956, 962, 964, 978, 871, 973, 990, 1010, 1025,1034, or 1098, or an amino acid sequence at least 80% (such as at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%) identical to any one of SEQ ID NOs: 856, 872, 881, 888, 902, 908,917, 924, 930, 933, 937, 938, 940, 953, 956, 962, 964, 978, 871, 973,990, 1010, 1025, 1034, or 1098.

Interface Residues

In more embodiments, the gp120 portion of the recombinant HIV-1 Envectodomain (from the heterologous HIV-1 strain) can further include oneor more substitutions at the gp120-gp41 interface that introduceresidues from the first HIV-1 strain. In some embodiments, suchsubstitutions can enhance the stabilization of the HIV-1 ectodomain inthe prefusion mature conformation by maintaining native BG505interaction between the membrane proximal “platform” and the core gp120.In some embodiments, the substitutions at the gp120-gp41 interface thatintroduce residues from the first HIV-1 strain can include substitutionsat gp120 positions 46-54, 70-75, 84-89, 99, 102, 106, 107, 114, 215,220-224, 226, 244, 471-473, and 476-477 (referred to herein as“Interface Residue Set A”). In some embodiments, the recombinant HIV-1Env ectodomain includes gp120 residues 31-45 and 478-507, and gp41residues (e.g., 512-664), from a first HIV-1 strain (such as the BG505strain), and includes gp120 residues 46-477 from a heterologous strainof HIV-1 (such as the CAP256.SU, BB201.B42, KER2018.11, CH070.1,ZM233.6, Q23.17, A244, WITO.33, JRFL, 426c (with N276D, N460D, N463D),d45-01dG5, B1369.9A, MB201.A1, QH209.14M.A2, 0921.V2.C14, 16055-2.3,25925-2.22, 286.36, CAP45.G3, DU156.12, DU422.01, MW965.26, ZM53.12,ZM55.28a, ZM106.9, 3301.V1.C24, 6545.V4.C1, 620345.c1, C1080.c3,C4118.09, CNE55, TH966.8, AC10.29, CH038.12, CNE58, or CH117.4 strain ofHIV-1), and further includes substitutions in the gp120 residues fromthe heterologous strain that introduce residues from the first HIV-1strain at the Interface Residue Set A positions of gp120. Non-limitingexamples of sequences of such chimeric HIV-1 Env ectodomains (that mayalso include one or more amino acid substitutions, such as 201C/433C andSOSIP substitutions to stabilize the HIV-1 ectodomain in the prefusionmature closed conformation) are provided herein as SEQ ID NOs: 579-586,588-595, and 1036-1056. Thus, in some embodiments, the recombinant HIV-1Env protein includes an amino acid sequence set forth as any one of SEQID NOs: 579-586, 588-595, and 1036-1056, or an amino acid sequence atleast 80% (such as at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs:579-586, 588-595, and 1036-1056.

Additional Description of Chimeric Domains

In some embodiments, the chimeric HIV-1 Env ectodomain can furtherinclude additional structural domains or elements from the first HIV-1strain (such as BG505) in place of those of the heterologous strain, forexample, strand C of the V1V2 domain (such as gp120 positions 166-173),a V3 domain (such as gp120 positions 296-331), a V2 loop (such as gp120positions 154-205), a V1 loop (such as gp120 positions 119-153),positions 191-205. In some embodiments, the chimeric HIV-1 Envectodomain can include from the first HIV-1 strain (such as BG505): a V2loop and a V3 loop; a Strand C of the V1V2 domain and a V3 domain;positions 191-205 and a Strand C of the V1V2 domain; a V1 loop and a V3domain; a V1 loop, a Strand C of the V1V2 domain, and a V3 domain; a V1loop, a V2 loop, and a V3 domain; or a V1V2 domain. Exemplary sequencesconcerning such chimeric HIV-1 Env ectodomains are provided as SEQ IDNOs: 1727-1764.

Chimeras of Three Strains

In additional embodiments, the recombinant HIV-1 Env ectodomain trimercan be a chimera having unique antigenic characteristics that providefor binding to mature and unmutated common ancestor (UCA) forms ofmultiple classes of broadly neutralizing antibodies (e.g., targeting theCD4 binding site and the V1V2 domain). Such recombinant HIV-1 Envectodomain trimers are of particular interest for use as a “prime”immunogen in a prime-boost immunization protocol for eliciting an immuneresponse to HIV-1 Env.

For example, in some embodiments, the recombinant HIV-1 Env ectodomaintrimer can be a chimera comprising amino acid sequences from three HIV-1strains, including a membrane proximal “platform” from a first strain, aV1V2 domain from a second strain, and the remainder from a heterologousstrain. In a non-limiting example, the V1V2 domain can be from an Envprotein (such as one of CAP256.SU, BB201.B42, KER2018.11, CH070.1,ZM233.6, Q23.17, A244, T250-4, or WITO.33) that binds to a UCA form of abroadly neutralizing antibody (e.g., VRC26 or PGT145), such as describedin Example 13. The remainder sequences of the chimera can also be froman Env protein that binds to a UCA form of a broadly neutralizingantibody (such as 45_01dG5 or 426c with amino acid substitutions toremove N-linked glycan sequons at positions 276, 460, 463), such as aUCA form or a VRC01-class antibody, for example VRC01 gHgL as describedin Example 13. The sequences of the first, second, and heterologousstrains are further modified to include the one or more amino acidsubstitutions that stabilize the recombinant HIV-1 Env ectodomain trimerin the prefusion mature closed conformation (such as SOS, IP, and DSsubstitutions), and can also include additional substitutions as needed,for example, substitutions to increase protease cleavage (such as the R6substitution), or to increase or decrease the desired number of glycans(such as addition of glycan sequons at positions 504 and 661, and/or atposition 332).

In some embodiments, the recombinant HIV-1 Env ectodomain trimer can bea chimera comprising amino acid sequences from three HIV-1 strains,wherein the recombinant HIV-1 Env ectodomain includes (1) a gp41ectodomain (such as positions 512-664), an N-terminal region of thegp120 polypeptide comprising a β-4 strand, and a C-terminal region ofthe gp120 polypeptide comprising a β26 strand, from a first strain ofHIV-1 (such as BG505), (2) a V1V2 domain (such as gp120 positions126-196) of the gp120 polypeptide from a second strain of HIV-1 (such asone of CAP256.SU, BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244,T250-4, or WITO.33; and (3) the remaining sequence of the gp120polypeptide from a heterologous strain of HIV-1 (such as 45_01dG5 or426c with amino acid substitutions to remove N-linked glycan sequons atpositions 276, 460, 463). In some such embodiments, the N-terminalregion of the gp120 polypeptide can further comprises a β-3 strand fromthe first HIV-1 strain; and the C-terminal region of the gp120polypeptide further comprises a β25 strand or a β25 strand and a α5helix from the first HIV-1 strain. In additional embodiments, the N- andC-terminal regions of the gp120 polypeptide comprise gp120 positions31-45 and 478-508, respectively. the gp120 polypeptide can furthercomprises positions 46-54, 70-75, 84-89, 99, 102, 106, 107, 114, 215,220-224, 226, 244, 471-473, and 476-477 from the first HIV-1 strain. Thesequences of the first, second, and heterologous strains are furthermodified to comprise the one or more amino acid substitutions thatstabilize the recombinant HIV-1 Env ectodomain trimer in the prefusionmature closed conformation.

In some embodiments, the second and heterologous strains arerespectively one of: CAP256.SU and 426c; BB201.B42 and 426c; KER2018.11and 426c; CH070.1 and 426c; ZM233.6 and 426c; Q23.17 and 426c; A244 and426c; T250-4 and 426c; WITO.33 and 426c; CAP256.SU and 45_O1dG5;BB201.B42 and 45_01dG5; KER2018.11 and 45_01dG5; CH070.1 and 45_01dG5;ZM233.6 and 45_01dG5; Q23.17 and 45_01dG5; A244 and 45_01dG5; T250-4 and45_01dG5; or WITO.33 and 45_01dG5; and wherein the 426c strain furthercomprises amino acid substitutions to remove the N-linked glycan sequonsat positions 276, 460, 463. The sequences of the first, second, andheterologous strains are further modified to include the one or moreamino acid substitutions that stabilize the recombinant HIV-1 Envectodomain trimer in the prefusion mature closed conformation (such asSOS, IP, and DS substitutions), and can also include additionalsubstitutions as needed,

In some embodiments, the second HIV-1 strain (providing the V1V2 domain)can be one of B1369.9A, MB201.A1, QH209.14M.A2, 0921.V2.C14, 16055-2.3,25925-2.22, 286.36, CAP45.G3, DU156.12, DU422.01, MW965.26, ZM53.12,ZM55.28a, ZM106.9, 3301.V1.C24, 6545.V4.C1, 620345.c1, C1080.c3,C4118.09, CNE55, TH966.8, AC10.29, CH038.12, CNE58, CH117.4, CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, WITO.33,or JRFL. For example, the second HIV-1 strain can be one of CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, WITO.33,and JRFL.

Non-limiting examples of sequences of such chimeric HIV-1 Envectodomains (that may also include one or more amino acid substitutions,such as 201C/433C and SOSIP substitutions to stabilize the HIV-1ectodomain in the prefusion mature closed conformation) are providedherein as SEQ ID NOs: 2146-2159. Thus, in some embodiments, therecombinant HIV-1 Env protein includes an amino acid sequence set forthas any one of SEQ ID NOs: 2146, 2147, 2148, 2149, 2150, 2151, 2152,2153, 2154, 2155, 2156, 2157, 2158, or 2159, or an amino acid sequenceat least 80% (such as at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs:2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157,2158, or 2159.

Residue Set B

In more embodiments, the gp120 portion of the recombinant HIV-1 Envectodomain (from the heterologous HIV-1 strain) can include additionalsubstitutions to alter the antigenicity of the ectodomain. In someembodiments, the substitutions at the gp120-gp41 interface thatintroduce residues from the first HIV-1 strain can include substitutionsat gp120 positions 133-134, 164, 169, 308, and 316 (referred to hereinas “Residue Set B”). In some embodiments, the recombinant HIV-1 Envectodomain includes gp120 residues 31-45 and 478-507, and gp41 residues(e.g., 512-664), from a first HIV-1 strain (such as the BG505 strainwith SOSIP substitutions set forth as SEQ ID NO: 3), and includes gp120residues 46-477 from a heterologous strain of HIV-1 (such as theCAP256.SU, BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244,WITO.33, B1369.9A, MB201.A1, QH209.14M.A2, 0921.V2.C14, 16055-2.3,25925-2.22, 286.36, CAP45.G3, CNE58, DU156.12, DU422.01, MW965.26,ZM53.12, ZM55.28a, ZM106.9, 3301.V1.C24, 6545.V4.C1, 620345.c1,C1080.c3, C4118.09, CNE55, TH966.8, AC10.29, CH038.12, or CH117.4 strainof HIV-1), and further includes substitutions in the gp120 residues fromthe heterologous strain that introduce residues from the first HIV-1strain at the Residue Set B positions of gp120. Non-limiting examples ofsequences of such chimeric HIV-1 Env ectodomains (that may also includeone or more amino acid substitutions, such as 201C/433C and SOSIPsubstitutions to stabilize the HIV-1 ectodomain in the prefusion matureclosed conformation) are provided herein as SEQ ID NOs: 1114-1142. Thus,in some embodiments, the recombinant HIV-1 Env protein includes an aminoacid sequence set forth as any one of SEQ ID NOs: 1114-1142, or an aminoacid sequence at least 80% (such as at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) identical to any oneof SEQ ID NOs: 1114-1142.

Residue Set C

In more embodiments, the gp120 portion of the recombinant HIV-1 Envectodomain (from the heterologous HIV-1 strain) can include additionalsubstitutions to alter the antigenicity of the ectodomain. In someembodiments, the substitutions at the gp120-gp41 interface thatintroduce residues from the first HIV-1 strain can include substitutionsat gp120 positions 49, 133-134, 149-152, 164, 169, 188, 190, 211, 223,252, 281, 293, 308, 316, 336, 340, 352, 360, 362-363, 369, 372, 393,410, 432, 442, 444, 446, 474, and 476 (referred to herein as “ResidueSet C”). In some embodiments, the recombinant HIV-1 Env ectodomainincludes gp120 residues 31-45 and 478-507, and gp41 residues (e.g.,512-664), from a first HIV-1 strain (such as the BG505 strain with SOSIPsubstitutions set forth as SEQ ID NO: 3), and includes gp120 residues46-477 from a heterologous strain of HIV-1 (such as the CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, WITO.33,B1369.9A, MB201.A1, QH209.14M.A2, 0921.V2.C14, 16055-2.3, 25925-2.22,286.36, CAP45.G3, CNE58, DU156.12, DU422.01, MW965.26, ZM53.12,ZM55.28a, ZM106.9, 3301.V1.C24, 6545.V4.C1, 620345.c1, C1080.c3,C4118.09, CNE55, TH966.8, AC10.29, CH038.12, or CH117.4 strain ofHIV-1), and further includes substitutions in the gp120 residues fromthe heterologous strain that introduce residues from the first HIV-1strain at the Residue Set C positions of gp120. Non-limiting examples ofsequences of such chimeric HIV-1 Env ectodomains (that may also includeone or more amino acid substitutions, such as 201C/433C and SOSIPsubstitutions to stabilize the HIV-1 ectodomain in the prefusion matureclosed conformation) are provided herein as SEQ ID NOs: 1143-1171. Thus,in some embodiments, the recombinant HIV-1 Env protein includes an aminoacid sequence set forth as any one of SEQ ID NOs: 1143-1171, or an aminoacid sequence at least 80% (such as at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) identical to any oneof SEQ ID NOs: 1143-1171.

Residue Set D

In more embodiments, the gp120 portion of the recombinant HIV-1 Envectodomain (from the heterologous HIV-1 strain) can include additionalsubstitutions to alter the antigenicity of the ectodomain. In someembodiments, the substitutions at the gp120-gp41 interface thatintroduce residues from the first HIV-1 strain can include substitutionsat gp120 positions of Residue Set C and also gp120 positions 46, 60,62-63, 84-85, 87, 99, 102, 130, 132, 135, 153, 158, 160-161, 165-167,171-173, 175, 177-178, 181, 184-185, 189, 202, 232, 234, 236, 240,268-271, 275, 277, 287, 289, 292, 295, 297, 305, 315, 317, 319, 322,328, 330, 332-335, 337, 339, 343-347, 350-351, 357, 371, 375, 379, 387,389, 394, 411, 412-413, 415, 424, 426, 429, 440, 460-461, 465, 475, and477 (referred to herein as “Residue Set D”). In some embodiments, therecombinant HIV-1 Env ectodomain includes gp120 residues 31-45 and478-507, and gp41 residues (e.g., 512-664), from a first HIV-1 strain(such as the BG505 strain with SOSIP substitutions set forth as SEQ IDNO: 3), and includes gp120 residues 46-477 from a heterologous strain ofHIV-1 (such as the CAP256.SU, BB201.B42, KER2018.11, CH070.1, ZM233.6,Q23.17, A244, WITO.33, BI369.9A, MB201.A1, QH209.14M.A2, 0921.V2.C14,16055-2.3, 25925-2.22, 286.36, CAP45.G3, CNE58, DU156.12, DU422.01,MW965.26, ZM53.12, ZM55.28a, ZM106.9, 3301.V1.C24, 6545.V4.C1,620345.c1, C1080.c3, C4118.09, CNE55, TH966.8, AC10.29, CH038.12, orCH117.4 strain of HIV-1), and further includes substitutions in thegp120 residues from the heterologous strain that introduce residues fromthe first HIV-1 strain at the Residue Set C and Residue Set D positionsof gp120. Non-limiting examples of sequences of such chimeric HIV-1 Envectodomains (that may also include one or more amino acid substitutions,such as 201C/433C and SOSIP substitutions to stabilize the HIV-1ectodomain in the prefusion mature closed conformation) are providedherein as SEQ ID NOs: 1172-1200. Thus, in some embodiments, therecombinant HIV-1 Env protein includes an amino acid sequence set forthas any one of SEQ ID NOs: 1172-1200, or an amino acid sequence at least80% (such as at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%) identical to any one of SEQ ID NOs:1172-1200.

Additional Substitutions

The chimeric recombinant HIV-1 Env ectodomain can be mutated to includeone or more of the disclosed amino acid substitutions to generate achimeric recombinant HIV Env protein (or fragment thereof, such as agp140 or gp145 protein) that is stabilized in a prefusion mature closedconformation. For example, in some non-limiting embodiments, cysteinesubstitutions at positions 201 and 433, and the SOSIP mutations, aremade to a recombinant HIV-1 Env ectodomain including a V1V2 domain froma CAP256.SU, BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244,T250-4, or WITO.33 strain of HIV-1 to generate the recombinant HIV-1 Envectodomain that can form a trimer stabilized in the prefusion matureclosed conformation. In some embodiments, the N-terminal residue of thetransplanted V1V2 domain can include one of Env positions 120-130 andthe C-terminal residue of the transplanted V1V2 domain can include oneof Env positions 195-205. In one non-limiting example, the transplantedV1V2 domain includes HIV-1 Env positions 126-196. Exemplary sequences ofHIV-1 ectodomains including a transplanted V1V2 domain are providedunder code H of the Table in Table 13 and as SEQ ID NOs: 379-386, or511-518.

Sequences from additional HIV-1 strains (such as a third, fourth orfifth strain) can be incorporated into the chimeric HIV-1 Envectodomain. For example, any of the chimeric HIV-1 Env ectodomainsdisclosed herein can be further modified to include all or portions ofthe V1V2 domain (such as strand C of the V1V2 domain, for example, HIV-1Env positions 166-173) from a heterologous HIV-1 strain (such as CAP256SU). In a non-limiting embodiment, SEQ ID NO: 382(CNE58_SU-strandC_bg505-NCgp120+gp41.SOSIP) provided herein includesgp41 and gp120 N- and C-terminal regions (31-45 and 478-507,respectively) from BG505.SOSIP.664, with residues 166-173 (V1V2 strandC) from CAP256 SU, and the rest of gp120 from CNE58.

Any of the chimeric HIV-1 Env ectodomain trimers provided herein cancomprise gp120-gp41 protomers that are single chain Env ectodomains,including a single polypeptide chain including the gp120 polypeptidelinked to the gp41 ectodomain by a heterologous peptide linker.

In some embodiments, the gp120/gp41 protomers in the chimeric HIV-1 Envectodomain can comprise an amino acid sequence set forth as any one ofSEQ ID NOs: 1580-1610, 1648-1650, 1657-1659, 1663-1673, 1676-2098, or anamino acid sequence at least 90% identical thereto.

In any of the disclosed embodiments that include a chimeric V1V2 domain,the V1V2 domain can comprise or consist of gp120 positions 126-196 (HXB2numbering).

Single Chain HIV-1 Env Proteins

In some embodiments, the recombinant HIV-1 Env ectodomain is a singlechain HIV-1 Env protein, which includes a single polypeptide chainincluding the gp120 polypeptide and the gp41 ectodomain. Native HIV-1Env sequences include a furin cleavage site at position 511 (e.g.,REKR₅₁₁), which is cleaved by a cellular protease to generate the gp120and gp41 polypeptides. The disclosed single chain proteins do notinclude the furin cleavage site separating the gp120 and gp41polypeptides; therefore, when produced in cells, the Env polypeptide isnot cleaved into separate gp120 and gp41 polypeptides.

Single chain HIV-1 proteins can be generated by mutating the furincleavage site to prevent cleave and formation of separate gp120 and gp41polypeptide chains. In several embodiments, the gp120 and gp41polypeptides in the single chain HIV-1 Env protein are joined by alinker, such as a peptide linker. Examples of peptide linkers that canbe used include glycine, serine, and glycine-serine linkers, such as aG, S, GG, GS, SG, GGG, or GSG linker or any of the linkers set forth asSEQ ID NOs: 519-542. In some embodiments, the peptide liker can comprisea 10 amino acid glycine-serine peptide linker, such as a peptide linkercomprising the amino acid sequence set forth as SEQ ID NOs: 528(GGSGGGGSGG). In some embodiments, the single chain HIV-1 protein caninclude a heterologous peptide linker between one of HIV-1 Env residues507 and 512, 503 and 519, 504 and 519, 503 and 522, or 504 and 522. Insome embodiments, the single chain HIV-1 protein can include aheterologous peptide linker between HIV-1 Env residues 507 and 512.

Any amino acid substitution or insertion can be used that effectivelyprevents furin (or other protease) cleavage of HIV-1 Env into separategp120 and gp41 polypeptide chains, and also allows folding of the HIV-1Env ectodomain into its prefusion mature closed conformation.

Any of the stabilizing mutations (or combinations thereof) disclosedherein can be included in the single chain HIV-1 Env protein as long asthe single chain HIV-1 Env protein retains the HIV-1 Env prefusionmature closed conformation. For example, in some embodiments, the singlechain HIV-1 Env protein can include cysteine substitutions at positions201 and 433 that form a disulfide bond and one or more of the pairs ofcysteine substitutions listed in Table 9, or the single chain HIV-1 Envprotein can include cysteine substitutions at positions 201 and 433 thatform a disulfide bond and further include the SOSIP mutations.

It will be appreciated that the single chain HIV-1 Env proteins can beincorporated into any embodiment disclosed herein in which the cleavedHIV-1 Env proteins can be used. For example, the single chain HIV-1 Envproteins can be linked to a protein nanoparticle subunit to generate aprotein nanoparticle including the single chain Env protein, and canalso be used in the context of a chimeric HIV-1 Env ectodomain includingsequences from two or more different strains of HIV-1.

Exemplary single chain HIV-1 Env sequences are provided under code B ofthe Table in Table 13 and set forth as SEQ ID NOs: 210-408. Additionalexemplary single chain chimeric HIV-1 Env ectodomain sequences areindicated as such in column 7 of the tables in Table 13, and alsoprovides as SEQ ID NOs: 1078-1098, and 1643-1650.

Membrane Anchored Embodiments

In some embodiments, the recombinant HIV-1 Env ectodomain is a membraneanchored protein, for example, the recombinant Env ectodomain can belinked to a transmembrane domain. The transmembrane domain can be linkedto any portion of the recombinant HIV-1 Env ectodomain, as long as thepresence of the transmembrane domain does not disrupt the structure ofthe HIV-1 Env ectodomain, or its ability to induce an immune response toHIV-1. In non-limiting examples, the transmembrane domain can be linkedto the N- or C-terminal reside of a gp120 polypeptide, or the C-terminalresidue of a gp41 ectodomain included in the recombinant HIV-1 Envprotein. In some embodiments, the C-terminal residue of the gp41ectodomain included in the recombinant HIV-1 Env ectodomain can belinked to the transmembrane domain. One or more peptide linkers (such asa gly-ser linker, for example a 10 amino acid glycine-serine peptidelinker, such as a peptide linker comprising the amino acid sequence setforth as SEQ ID NOs: 528 (GGSGGGGSGG)) can be used to link thetransmembrane domain and the gp120 or gp41 protein. In some embodimentsa native HIV-1 Env MPER sequence can be used to link the transmembranedomain and the gp120 or gp41 protein.

Non-limiting examples of transmembrane domains for use with thedisclosed embodiments include the BG505™ domain(KIFIMIVGGLIGLRIVFAVLSVIHRVR, SEQ ID NO: 758), the Influenza AHemagglutinin™ domain (ILAIYSTVASSLVLLVSLGAISF, SEQ ID NO: 760, and theInfluenza A Neuraminidase TM domain (IITIGSICMVVGIISLILQIGNIISIWVS, SEQID NO: 762). Nucleic acid sequences encoding these TM domains areprovided as SEQ ID NOs: 759, 761, and 763, respectively.

The recombinant HIV-1 Env ectodomain linked to the transmembrane domaincan include any of the stabilizing mutations provided herein. Forexample, the transmembrane domain can be linked to the C-terminalresidue of a gp41 ectodomain included in a recombinant HIV-1 Envectodomain including the DS substitutions (I201C/A433C), and/or can belinked to any of the disclosed chimeric recombinant HIV-1 Envectodomains. Exemplary sequences of recombinant HIV-1 Env ectodomain (ora fragment thereof) linked to a transmembrane domain and including aminoacid substitutions to stabilize the ectodomain in the prefusion matureclosed conformation are provided under code T of the Table in Table 13,and as SEQ ID NOs: 544-571, and 1765-2098.

Linkage to a Trimerization Domain

In several embodiments, the recombinant HIV-1 Env ectodomain can belinked to a trimerization domain, for example the C-terminus of the gp41protein included in the recombinant HIV-1 Env ectodomain can be linkedto the trimerization domain. The trimerization domain can promotestrimerization of the three protomers of the recombinant HIV-1 Envprotein. Non-limiting examples of exogenous multimerization domains thatpromote stable trimers of soluble recombinant proteins include: the GCN4leucine zipper (Harbury et al. 1993 Science 262:1401-1407), thetrimerization motif from the lung surfactant protein (Hoppe et al. 1994FEBS Lett 344:191-195), collagen (McAlinden et al. 2003 J Biol Chem278:42200-42207), and the phage T4 fibritin Foldon (Miroshnikov et al.1998 Protein Eng 11:329-414), any of which can be linked to therecombinant HIV-1 Env ectodomain (e.g., by linkage to the C-terminus ofthe gp41 polypeptide to promote trimerization of the recombinant HIV-1protein, as long as the recombinant HIV-1 Env ectodomain retainsspecific binding activity for a mature closed conformation specificantibody, prefusion-specific antibody (e.g., PGT122), and/or includes aHIV-1 Env mature closed conformation.

In some examples, the recombinant HIV-1 Env ectodomain can be linked toa Foldon domain, for example, the recombinant HIV-1 Env ectodomain caninclude a gp41 polypeptide with a Foldon domain linked to itsC-terminus. In specific examples, the Foldon domain is a T4 fibritinFoldon domain such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTF(SEQ ID NO: 578), which adopts a β-propeller conformation, and can foldand trimerize in an autonomous way (Tao et al. 1997 Structure5:789-798). Modified Foldon domains can also be used, such as a Foldondomain including an amino acid sequence set forth asGYIPEAPRDGQCYVRCDGEWVLLSTF (SEQ ID NO: 752), GYIPECPRDGQAYVCKDGEWVLLSTF(SEQ ID NO: 753), GYIPEAPRDGQCYCRKDGEWVLLSTF (SEQ ID NO: 754), orGYIPEAPRDGQACVRKDGECVLLSTF (SEQ ID NO: 755). These modified Foldondomains include amino acid substitutions that add two cysteine residuesfor formation of stabilizing disulfide bonds. In some embodiments, anyof the disclosed recombinant HIV-1 Env ectodomains can be linked to amodified Foldon domain as described herein.

Exemplary sequences of recombinant HIV-1 Env ectodomain linked to atrimerization domain are provided under code G of the Table in Table 13,and as SEQ ID NOs: 508-510.

Typically, the heterologous trimerization domain is positionedC-terminal to the gp41 polypeptide. Optionally, the multimerizationdomain is connected to the recombinant HIV-1 Env ectodomain via alinker, such as an amino acid linker. Exemplary linkers are providedherein and are known in the art; non-limiting examples include Gly orGly-Ser linkers, such as the amino acid sequence: GGSGGSGGS; SEQ ID NO:574). Numerous conformationally neutral linkers are known in the artthat can be used in this context without disrupting the conformation ofthe recombinant HIV-1 Env protein. Some embodiments include a proteasecleavage site for removing the trimerization domain from the HIVpolypeptide, such as, but not limited to, a thrombin site between therecombinant HIV-1 Env ectodomain and the trimerization domain.

Additional Descriptions of Recombinant HIV-1 Env Ectodomains

Any of the recombinant HIV-1 Env ectodomains disclosed herein canfurther include an N-linked glycosylation site at gp120 position 332 (ifnot already present on the ectodomain). For example, by T332Nsubstitution in the case of BG505 based immunogens. The presence of theglycosylation site at N332 allows for binding by 2G12 antibody.

Any of the recombinant HIV-1 Env ectodomains disclosed herein caninclude a lysine residue at gp120 position 168 (if not already presenton the ectodomain). For example, the lysine residue can be added byamino acid substitution (such as an E168K substitution in the case ofthe JR-FL based immunogens). The presence of the lysine residue atposition 168 allows for binding of particular broadly neutralizingantibodies to the V1V2 loop of gp120.

Any of the recombinant HIV-1 Env ectodomains disclosed herein caninclude an arginine residue at gp120 position 368 (if not alreadypresent on the ectodomain). For example, the arginine residue can beadded by amino acid substitution (such as a D368R substitution). Thepresence of the arginine residue at position 368 reduces binding of CD4to the HIV-1 Env ectodomain to inhibit the trimer from adopting theCD4-bound conformation.

Any of the recombinant HIV-1 Env ectodomains disclosed herein canfurther include a non-natural disulfide bond between gp120 positions 201and 433 (if not already present on the ectodomain). For example, thenon-natural disulfide bond can be introduced by including cysteinesubstitutions at positions 201 and 433. The presence of the non-naturaldisulfide bond between residues 201 and 433 contributes to thestabilization of the HIV-1 Env ectodomain in the prefusion mature closedconformation.

Any of the recombinant HIV-1 Env ectodomains disclosed herein canfurther include a non-natural disulfide bond between HIV-1 Env positions501 and 605 (if not already present on the ectodomain). For example, thenon-natural disulfide bond can be introduced by including cysteinesubstitutions at positions 501 and 605. The presence of the non-naturaldisulfide bond between positions 501 and 605 contributes to thestabilization of the HIV-1 Env ectodomain in the prefusion mature closedconformation.

Any of the recombinant HIV-1 Env ectodomains disclosed herein canfurther include a proline residue at HIV-1 Env positions 559 (if notalready present on the ectodomain). For example, the proline residue canbe introduced at position 559 by amino acid substitution (such as anI559P substitution). The presence of the proline residue at position 559contributes to the stabilization of the HIV-1 Env ectodomain in theprefusion mature closed conformation.

Any of the recombinant HIV-1 Env ectodomains disclosed herein canfurther include a non-natural disulfide bond between HIV-1 Env positions501 and 605 and a proline residue at HIV-1 Env positions 559 (if notalready present on the ectodomain). For example, the non-naturaldisulfide bond can be introduced by including cysteine substitutions atpositions 501 and 605, and the proline residue can be introduced atposition 559 by amino acid substitution (such as an I559P substitution).The presence of the non-natural disulfide bond between positions 501 and605 and the proline residue at position 559 contributes to thestabilization of the HIV-1 Env ectodomain in the prefusion mature closedconformation.

Any of the recombinant HIV-1 Env ectodomains disclosed herein can befurther modified to be a singly chain HIV-1 Env ectodomain including a10 amino acid glycine serine linker between HIV-1 Env residues 507 and512 (if the recombinant HIV-1 Env ectodomains is not already a singlechain ectodomain).

Any of the recombinant HIV-1 Env ectodomains disclosed herein can befurther modified to include the “R6” mutation, which provides sixArginine residues in place of the naïve furin cleavage site betweengp120 and gp41.

Any of the soluble recombinant HIV-1 Env ectodomain trimers disclosedherein can include mutations to add a N-linked glycan sequon at position504, position 661, or positions 504 and 661, to increase glycosylationof the membrane proximal region of the ectodomain.

Any of the recombinant HIV-1 Env ectodomain trimers disclosed hereinthat include a protease cleavage site between the gp120 and gp41polypeptides can be modified to be a single chain HIV-1 Env ectodomainby mutation of the protease cleavage site for example by introducing a10 amino acid linker connecting gp120 and gp41 or a 15 amino acid linkerconnecting g120 and gp41, for example as shown in SEQ ID NOs: 2158 (15AA linker) and 2159 (10 AA linker).

In some embodiments, the recombinant HIV-1 Env ectodomain can comprise acircular permutant of the Env ectodomain. For example, the circularpermutant can comprise, from N-terminus to C-terminus,

(A) the gp41 polypeptide and the gp120 polypeptide linked by a peptidelinker or directly linked; or

(B) a first segment of the gp41 polypeptide comprising a α6 helix, a α7helix, and/or a β27 strand of the prefusion mature closed conformationof the HIV-1 Env protein;

the gp120 polypeptide; and

a second segment of the gp41 polypeptide comprising the α8 helix and/orthe α9 helix of the prefusion mature closed conformation, wherein

the first and second segments of the gp41 polypeptide are linked to thegp120 polypeptide by a peptide linker, or are directly linked to thegp120 polypeptide.

The recombinant HIV-1 Env ectodomain comprising the circular permutantof the Env ectodomain can further comprise any of the amino acidsubstitutions disclosed herein for stabilizing the HIV-1 Env ectodomainin the prefusion mature closed conformation, such as the DS-SOSIPsubstitutions.

C. V1V2V3 Immunogens

The V1, V2, and V3 domains of HIV-1 Env are located on the apex of thetrimer in the prefusion mature closed conformation and include regionsrecognized by several neutralizing antibodies. Provided herein areimmunogens that include these minimal domains of the HIV-1 Env proteinin a format that maintains their structure in the prefusion matureclosed conformation, and which are useful, for example, for inducing animmune response to HIV-1 Env. These immunogens are also useful forspecific binding to antibodies that target the V1, V2, or V3 domains ofHIV-1 Env, for example as probes to identify or detect such antibodies.

In several embodiments, the V1, V2, and V3 domains are included on aprotein scaffold, such as a scaffold protein based on the 1VH8 protein(SEQ ID NO: 855), which is deposited in the Protein Data Bank as No.1VH8, and incorporated by reference herein in its entirety. In anotherexample, the “scaffold” can be any of the recombinant HIV-1 ectodomaintrimers described herein, and the V1V2V3 immunogen can be included onthe recombinant HIV-1 ectodomain trimer in place of the correspondingsequence of the HIV-1 ectodomain (e.g., the V1V2V3 immunogen can be“transplanted” on the recombinant HIV-1 ectodomain trimer). In someembodiments, the V1V2V3 scaffold protein comprises a circular permutantof the V1, V2, and V3 domains of HIV-1 linked to the 1VH8 protein. Insome embodiments, the V1V2V3 scaffold protein comprises from N- toC-terminus:

-   -   (1VH8 residues 36-159)-L₁-(1VH8 residues 2-15)-L₂-(V1V2        domain)-L₃-(V3 domain)        In some embodiments, the V1V2 domain portion of the V1V2V3        scaffold protein can include HIV-1 Env residues 120-203. In some        embodiments, the V3 domain portion of the V1V2V3 scaffold        protein can be a circular permutant of the V3 domain including,        from N- to C-terminus,    -   (HIV-1 Env residues 317-330)-L₄-(HIV-1 Env residues 297-314)        The linkers in the V1V2V3 scaffold protein are peptide linkers,        for example glycine-serine linkers. In some embodiments, the L₁        linker can include a GSG sequence, the L₂ linker can include SEQ        ID NO: 528 or SEQ ID NO: 854, the L₃ linker can include SEQ ID        NO: 319, and/or the L₄ linker can include AA-GSG-A.

Exemplary V1V2V3 scaffold proteins are provided as SEQ ID NOs: 836-843.In some embodiments, the immunogen includes an amino acid sequence atleast 80% (such as at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs:836-843.

The V1V2V3 scaffold protein can include any of the stabilizing mutationsdescribed herein that include mutations to the V1, V2, and/or V3 domainto stabilize the V1, V2, and/or V3 domains in the prefusion matureclosed conformation of the HIV-1 Env protein, for example the V1V2V3scaffold protein can include cysteine substitutions at positions 174 and319, or 175 and 320, which stabilize the V1, V2 and/or V3 domains in theprefusion mature closed conformation.

D. Protein Nanoparticles

In some embodiments a protein nanoparticle is provided that includes oneor more of the disclosed recombinant HIV-1 Env ectodomains stabilized ina prefusion mature closed conformation, or an immunogenic fragmentthereof. Such a protein nanoparticle can be specifically bound by one ormore antibodies that specifically bind to the HIV-1 Env prefusion matureclosed conformation, such as VRC26, PGT122, PGT145, and 35O22.Additionally, in several embodiments, the disclosed nanoparticles do notspecifically bind to an antibody that specifically binds to HIV-1 Env inits CD4 bound conformation, but not to HIV-1 Env in its prefusion matureclosed conformation. For example, the disclosed protein nanoparticles donot specifically bind to 17b antibody in the presence of a molar excessof CD4. Non-limiting example of nanoparticles include ferritinnanoparticles, an encapsulin nanoparticles, Sulfur Oxygenase Reductase(SOR) nanoparticles, and lumazine synthase nanoparticles, which arecomprised of an assembly of monomeric subunits including ferritinproteins, encapsulin proteins, SOR proteins, and lumazine synthaserespectively. Exemplary sequences of recombinant HIV-1 Env ectodomainslinked to a nanoparticle subunit are provided under code F in the Tableincluded in Table 13. To construct protein nanoparticles including aHIV-1 Env proteins stabilized in a prefusion mature closed conformationor immunogenic fragment thereof, the HIV-1 Env protein or fragment canbe linked to a subunit of the protein nanoparticle (such as a ferritinprotein, an encapsulin protein, a SOR protein, or a lumazine synthaseprotein). The fusion protein self-assembles into a nanoparticle underappropriate conditions.

In several embodiments, the protein nanoparticle comprises two or moreof the recombinant HIV-1 Env proteins, wherein the two or morerecombinant HIV-1 Env proteins are from at least two different strainsof HIV-1.

In some embodiments, the immunogen comprises a recombinant HIV-1 Envprotein linked to a protein nanoparticle subunit, and comprises an aminoacid sequence at least 80% (such as at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) identical to thesequence set forth as one of 471-507, or 596-645, wherein therecombinant HIV-1 Env protein linked to the nanoparticle subunit canoligomerizes to form a functional protein nanoparticle including arecombinant HIV-1 Env ectodomain trimer (or immunogenic fragmentthereof) in a prefusion mature closed conformation.

In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a ferritin subunit to construct aferritin nanoparticle. Ferritin nanoparticles and their use forimmunization purposes (e.g., for immunization against influenzaantigens) have been disclosed in the art (see, e.g., Kanekiyo et al.,Nature, 499:102-106, 2013, incorporated by reference herein in itsentirety). Ferritin is a globular protein that is found in all animals,bacteria, and plants, and which acts primarily to control the rate andlocation of polynuclear Fe(III)₂O₃ formation through the transportationof hydrated iron ions and protons to and from a mineralized core. Theglobular form of the ferritin nanoparticle is made up of monomericsubunits, which are polypeptides having a molecule weight ofapproximately 17-20 kDa. An example of the amino acid sequence of onesuch monomeric subunit is represented by SEQ ID NO: 575.

Each monomeric subunit has the topology of a helix bundle which includesa four antiparallel helix motif, with a fifth shorter helix (thec-terminal helix) lying roughly perpendicular to the long axis of the 4helix bundle. According to convention, the helices are labeled ‘A, B, C,D & E’ from the N-terminus respectively. The N-terminal sequence liesadjacent to the capsid three-fold axis and extends to the surface, whilethe E helices pack together at the four-fold axis with the C-terminusextending into the capsid core. The consequence of this packing createstwo pores on the capsid surface. It is expected that one or both ofthese pores represent the point by which the hydrated iron diffuses intoand out of the capsid. Following production, these monomeric subunitproteins self-assemble into the globular ferritin protein. Thus, theglobular form of ferritin comprises 24 monomeric, subunit proteins, andhas a capsid-like structure having 432 symmetry. Methods of constructingferritin nanoparticles are known to the person of ordinary skill in theart and are further described herein (see, e.g., Zhang, Int. J. Mol.Sci., 12:5406-5421, 2011, which is incorporated herein by reference inits entirety).

In specific examples, the ferritin polypeptide is E. coli ferritin,Helicobacter pylori ferritin, human light chain ferritin, bullfrogferritin or a hybrid thereof, such as E. coli-human hybrid ferritin, E.coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin.Exemplary amino acid sequences of ferritin polypeptides and nucleic acidsequences encoding ferritin polypeptides for use to make a ferritinnanoparticle including a recombinant HIV-1 Env ectodomain or immunogenicfragment thereof can be found in GENBANK®, for example at accessionnumbers ZP_03085328, ZP_06990637, EJB64322.1, AAA35832, NP_000137AAA49532, AAA49525, AAA49524 and AAA49523, which are specificallyincorporated by reference herein in their entirety as available Jun. 20,2014. In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a ferritin subunit including an aminoacid sequence at least 80% (such as at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) identical to aminoacid sequence set forth as SEQ ID NO: 575.

Non-limiting examples of a recombinant HIV-1 Env ectodomains stabilizedin a prefusion mature closed conformation or immunogenic fragmentsthereof linked to a ferritin subunit include the amino acid sequence setforth as any one of SEQ ID NO: 471, 473-475, 626-637, 797-802, 809-814,821-835, 1099-1113, and 1201-1218.

In additional embodiments, any of the disclosed recombinant HIV-1 Envproteins stabilized in a prefusion mature closed conformation orimmunogenic fragments thereof can be linked to a lumazine synthasesubunit to construct a lumazine synthase nanoparticle. The globular formof lumazine synthase nanoparticle is made up of monomeric subunits; anexample of the sequence of one such monomeric subunit is provides as theamino acid sequence set forth as SEQ ID NO: 576.

In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a lumazine synthase subunit includingan amino acid sequence at least 80% (such as at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%) identical toamino acid sequence set forth as SEQ ID NO: 576. Specific examples of arecombinant HIV-1 Env ectodomain stabilized in a prefusion mature closedconformation or immunogenic fragments thereof linked to a lumazinesynthase subunit is provided as the amino acid sequence set forth as SEQID NO: 472, 476-477, 638-645, 803-808, and 815-820.

In additional embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to an encapsulin subunit to construct anencapsulin nanoparticle. The globular form of the encapsulinnanoparticle is made up of monomeric subunits; an example of thesequence of one such monomeric subunit is provides as the amino acidsequence set forth as SEQ ID NO: 756.

In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to an encapsulin subunit including anamino acid sequence at least 80% (such as at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%) identical toamino acid sequence set forth as SEQ ID NO: 756.

Encapsulin proteins are a conserved family of bacterial proteins alsoknown as linocin-like proteins that form large protein assemblies thatfunction as a minimal compartment to package enzymes. The encapsulinassembly is made up of monomeric subunits, which are polypeptides havinga molecule weight of approximately 30 kDa. Following production, themonomeric subunits self-assemble into the globular encapsulin assemblyincluding 60 monomeric subunits. Methods of constructing encapsulinnanoparticles are known to the person of ordinary skill in the art, andfurther described herein (see, for example, Sutter et al., NatureStruct. and Mol. Biol., 15:939-947, 2008, which is incorporated byreference herein in its entirety). In specific examples, the encapsulinpolypeptide is bacterial encapsulin, such as E. coli or Thermotogamaritime encapsulin.

In additional embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a Sulfer Oxygenase Reductase (SOR)subunit to construct a recombinant SOR nanoparticle. In someembodiments, the SOR subunit can include the amino acid sequence setforth as SEQ ID NO: 577.

In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a SOR subunit including an amino acidsequence at least 80% (such as at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99%) identical to amino acidsequence set forth as SEQ ID NO: 577.

SOR proteins are microbial proteins (for example from thethermoacidophilic archaeon Acidianus ambivalens that form 24 subunitprotein assemblies. Methods of constructing SOR nanoparticles are knownto the person of ordinary skill in the art (see, e.g., Urich et al,Science, 311:996-1000, 2006, which is incorporated by reference hereinin its entirety). An example of an amino acid sequence of a SOR proteinfor use to make SOR nanoparticles is set forth in Urich et al., Science,311:996-1000, 2006, which is incorporated by reference herein in itsentirety.

In some examples, the disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to the N- or C-terminus, or placed withinan internal loop of a ferritin, encapsulin, SOR, or lumazine synthasesubunit, for example with a linker, such as a Ser-Gly linker. When theconstructs have been made in HEK 293 Freestyle cells, the fusionproteins are secreted from the cells and self-assembled intonanoparticles. The nanoparticles can be purified using known techniques,for example by a few different chromatography procedures, e.g. Mono Q(anion exchange) followed by size exclusion (SUPEROSE® 6)chromatography.

Several embodiments include a monomeric subunit of a ferritin,encapsulin, SOR, or lumazine synthase protein, or any portion thereofwhich is capable of directing self-assembly of monomeric subunits intothe globular form of the protein. Amino acid sequences from monomericsubunits of any known ferritin, encapsulin, SOR, or lumazine synthaseprotein can be used to produce fusion proteins with the disclosedrecombinant HIV-1 Env ectodomain or immunogenic fragment thereof, solong as the monomeric subunit is capable of self-assembling into ananoparticle displaying recombinant HIV-1 Env ectodomain or immunogenicfragment thereof on its surface.

The fusion proteins need not comprise the full-length sequence of amonomeric subunit polypeptide of a ferritin, encapsulin, SOR, orlumazine synthase protein. Portions, or regions, of the monomericsubunit polypeptide can be utilized so long as the portion comprisesamino acid sequences that direct self-assembly of monomeric subunitsinto the globular form of the protein.

In some embodiments, it may be useful to engineer mutations into theamino acid sequence of the monomeric ferritin, encapsulin, SOR, orlumazine synthase subunits. For example, it may be useful to alter sitessuch as enzyme recognition sites or glycosylation sites in order to givethe fusion protein beneficial properties (e.g., half-life).

It will be understood by those skilled in the art that fusion of any ofthe disclosed recombinant HIV-1 Env proteins stabilized in a prefusionmature closed conformation or immunogenic fragments thereof to theferritin, encapsulin, SOR, or lumazine synthase protein should be donesuch that the disclosed recombinant HIV-1 Env proteins stabilized in aprefusion mature closed conformation or immunogenic fragments thereofdoes not interfere with self-assembly of the monomeric ferritin,encapsulin, SOR, or lumazine synthase subunits into the globularprotein, and that the ferritin, encapsulin, SOR, or lumazine synthasesubunits do not interfere with the ability of the disclosed recombinantHIV-1 Env proteins stabilized in a prefusion mature closed conformationor immunogenic fragments thereof to elicit an immune response to HIV. Insome embodiments, the ferritin, encapsulin, SOR, or lumazine synthaseprotein and disclosed recombinant HIV-1 Env proteins stabilized in aprefusion mature closed conformation or immunogenic fragments thereofcan be joined together directly without affecting the activity of eitherportion. In other embodiments, the ferritin, encapsulin, SOR, orlumazine synthase protein and the disclosed recombinant HIV-1 Envproteins stabilized in a prefusion mature closed conformation orimmunogenic fragments thereof can be joined using a linker (alsoreferred to as a spacer) sequence. The linker sequence is designed toposition the ferritin, encapsulin, SOR, or lumazine synthase portion ofthe fusion protein and the recombinant HIV-1 Env ectodomain stabilizedin a prefusion mature closed conformation or immunogenic fragmentsthereof can be linked to an portion of the fusion protein, with regardto one another, such that the fusion protein maintains the ability toassemble into nanoparticles, and also elicit an immune response to HIV.In several embodiments, the linker sequences comprise amino acids.Preferable amino acids to use are those having small side chains and/orthose which are not charged. Such amino acids are less likely tointerfere with proper folding and activity of the fusion protein.Accordingly, preferred amino acids to use in linker sequences, eitheralone or in combination are serine, glycine and alanine. One example ofsuch a linker sequence is SGG. Amino acids can be added or subtracted asneeded. Those skilled in the art are capable of determining appropriatelinker sequences for construction of protein nanoparticles.

The disclosed recombinant HIV-1 Env proteins stabilized in a prefusionmature closed conformation or immunogenic fragments thereof can belinked to ferritin, encapsulin, SOR, or lumazine synthase subunits canself-assemble into multi-subunit protein nanoparticles, termed ferritinnanoparticles, encapsulin nanoparticles, SOR nanoparticles, and lumazinesynthase nanoparticles, respectively. The nanoparticles including adisclosed recombinant HIV-1 Env ectodomain stabilized in a prefusionmature closed conformation or immunogenic fragment thereof havesubstantially the same structural characteristics as the nativeferritin, encapsulin, SOR, or lumazine synthase nanoparticles that donot include the disclosed recombinant HIV-1 Env ectodomain orimmunogenic fragment thereof. That is, they contain 24, 60, 24, or 60subunits (respectively) and have similar corresponding symmetry.

Additional sequences of recombinant HIV-1 Env proteins as disclosedherein linked to a protein nanoparticle subunit are provided as SEQ IDNOs: 478-507.

In some embodiments, the recombinant HIV-1 Env ectodomain stabilized ina prefusion mature closed conformation or immunogenic fragment thereofcan be linked to Escherichia coli enzyme 2-hydroxypentadienoic acidhydratase subunit (see Montgomery et al, J. Mol. Biol. 396: 1379-1391,2010), which can include, for example, the amino acid sequence set forthas SEQ ID NO: 2101 or a fragment thereof. In some embodiments, adisclosed recombinant HIV-1 Env ectodomain stabilized in a prefusionmature closed conformation or immunogenic fragment thereof can be linkedto a cocksfoot mottle virus coat protein subunit (see Tars et al.,Virology 310: 287-297, 2003), which can include, for example, the aminoacid sequence set forth as SEQ ID NO: 2102 or a fragment thereof. Insome embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a Rice yellow mottle virus capsidprotein subunit (see Qu et al., Structure Fold. Des. 8: 1095-1103,2000), which can include, for example, the amino acid sequence set forthas SEQ ID NO: 2103 or a fragment thereof. In some embodiments, adisclosed recombinant HIV-1 Env ectodomain stabilized in a prefusionmature closed conformation or immunogenic fragment thereof can be linkedto a sesbania mosaic virus coat protein subunit (see Bhuvaneshwari etal., Structure 3: 1021-1030, 1995), which can include, for example, theamino acid sequence set forth as SEQ ID NO: 2104 or a fragment thereof.In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a tomato bushy stunt virus coatprotein subunit (see Hopper et al., J. Mol. Biol. 177: 701-713, 1984),which can include, for example, the amino acid sequence set forth as SEQID NO: 2105 or a fragment thereof. In some embodiments, a disclosedrecombinant HIV-1 Env ectodomain stabilized in a prefusion mature closedconformation or immunogenic fragment thereof can be linked to a phageMS2 protein capsid subunit (see van den Worm et al., Nucleic Acids Res.26: 1345-1351, 1998), which can include, for example, the amino acidsequence set forth as SEQ ID NO: 2106 or a fragment thereof. In someembodiments, a disclosed recombinant HIV-1 Env ectodomain stabilized ina prefusion mature closed conformation or immunogenic fragment thereofcan be linked to bacteriophage fr capsid subunit (see Liljas et al., J.Mol. Biol. 244: 279-290, 1994), which can include, for example, theamino acid sequence set forth as SEQ ID NO: 2107 or a fragment thereof.In some embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a bacteriophage phiCb5 coat proteinsubunit (see Plevka et al., J. Mol. Biol. 391: 635-647, 2009), which caninclude, for example, the amino acid sequence set forth as SEQ ID NO:2108 or a fragment thereof. In some embodiments, a disclosed recombinantHIV-1 Env ectodomain stabilized in a prefusion mature closedconformation or immunogenic fragment thereof can be linked to a HK97bacteriophage capsid subunit (see Helgstrand et al., J. Mol. Biol. 334:885, 2003), which can include, for example, the amino acid sequence setforth as SEQ ID NO: 2109 or a fragment thereof. In some embodiments, adisclosed recombinant HIV-1 Env ectodomain stabilized in a prefusionmature closed conformation or immunogenic fragment thereof can be linkedto a bacteriophage GA protein capsid subunit (see Tars et al., J. Mol.Biol. 271: 759-773, 1997), which can include, for example, the aminoacid sequence set forth as SEQ ID NO: 2110 or a fragment thereof. Insome embodiments, a disclosed recombinant HIV-1 Env ectodomainstabilized in a prefusion mature closed conformation or immunogenicfragment thereof can be linked to a bacteriophage PRR1 coat proteinsubunit (see Persson et al., J. Mol. Biol. 383: 914, 2008), which caninclude, for example, the amino acid sequence set forth as SEQ ID NO:2111 or a fragment thereof. In some embodiments, a disclosed recombinantHIV-1 Env ectodomain stabilized in a prefusion mature closedconformation or immunogenic fragment thereof can be linked to abacteriophage PP7 coat protein subunit (see Tars et al., ActaCrystallogr., Sect. D 56: 398, 2000), which can include, for example,the amino acid sequence set forth as SEQ ID NO: 2112 or a fragmentthereof. In some embodiments, a disclosed recombinant HIV-1 Envectodomain stabilized in a prefusion mature closed conformation orimmunogenic fragment thereof can be linked to a bacteriophage Q betacapsid subunit (see Golmohammadi et al., Structure 4: 543-554, 1996),which can include, for example, the amino acid sequence set forth as SEQID NO: 2113 or a fragment thereof.

E. Polynucleotides and Expression

Polynucleotides encoding a disclosed immunogen (e.g., a HIV-1 Envectodomain stabilized in a prefusion mature closed conformation, or animmunogenic fragment thereof), or protein nanoparticles (or a subunitthereof) or vectors, disclosed herein are also provided. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode theantigen. One of skill in the art can readily use the genetic code toconstruct a variety of functionally equivalent nucleic acids, such asnucleic acids which differ in sequence but which encode the sameantibody sequence, or encode a conjugate or fusion protein including thenucleic acid sequence.

In a non-limiting example, a polynucleotide sequence set forth as SEQ IDNO: 757, which encodes the single chain HIV-1 Env set forth as SEQ IDNO: 352. In another example, a polynucleotide sequence set forth as SEQID NO: 2119, which encodes the BG505.SOSIP.R6.664.T332N_I201C/A433CHIV-1 Env set forth as SEQ ID NO: 26. For reference, native BG505 DNAsequence is provided as SEQ ID NO: 2120.

In several embodiments, the nucleic acid molecule encodes a precursor ofa disclosed recombinant HIV-1 Env ectodomain or immunogenic fragmentthereof, that, when expressed in an appropriate cell, is processed intoa disclosed recombinant HIV-1 Env ectodomain or immunogenic fragmentthereof. For example, the nucleic acid molecule can encode a recombinantHIV-1 Env ectodomain including a N-terminal signal sequence for entryinto the cellular secretory system that is proteolytically cleaved inthe during processing of the HIV-1 Env protein in the cell. In someembodiments, the signal peptide includes the amino acid sequence setforth as residues 1-30 of SEQ ID NO: 2.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013). Product information from manufacturers ofbiological reagents and experimental equipment also provide usefulinformation. Such manufacturers include the SIGMA Chemical Company(Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The polynucleotides encoding a recombinant HIV-1 Env proteins stabilizedin a prefusion mature closed conformation, fragments thereof, andprotein nanoparticles (or a subunit thereof) can include a recombinantDNA which is incorporated into a vector into an autonomously replicatingplasmid or virus or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (such as a cDNA) independent ofother sequences. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

Polynucleotide sequences encoding recombinant HIV-1 Env proteinsstabilized in a prefusion mature closed conformation, fragments thereof,and protein nanoparticles (or a subunit thereof) can be operativelylinked to expression control sequences. An expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences. The expression control sequences include,but are not limited to, appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons.

DNA sequences encoding the recombinant HIV-1 Env proteins stabilized ina prefusion mature closed conformation, fragments thereof, and proteinnanoparticles (or a subunit thereof) can be expressed in vitro by DNAtransfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, e.g., Helgason andMiller (Eds.), 2012, Basic Cell Culture Protocols (Methods in MolecularBiology), 4^(th) Ed., Humana Press). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and W138, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression, desirable glycosylation patterns, or otherfeatures. In some embodiments, the host cells include HEK293 cells orderivatives thereof, such as GnTI⁴⁻ cells (ATCC® No. CRL-3022), orHEK-293F cells.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or viral vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a disclosedantigen, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, ViralExpression Vectors, Springer press, Muzyczka ed., 2011). One of skill inthe art can readily use an expression systems such as plasmids andvectors of use in producing proteins in cells including highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

In one non-limiting example, a disclosed immunogen is expressed usingthe pVRC8400 vector (described in Barouch et al., J. Virol, 79,8828-8834, 2005, which is incorporated by reference herein).

Modifications can be made to a nucleic acid encoding a recombinant HIV-1Env ectodomain stabilized in a prefusion mature closed conformation,fragment thereof, and protein nanoparticle (or a subunit thereof)described herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.

In addition to recombinant methods, the recombinant HIV-1 Env proteinsstabilized in a prefusion mature closed conformation, fragments thereof,and protein nanoparticles (or a subunit thereof) can also be constructedin whole or in part using protein synthesis methods known in the art.

F. Virus-Like Particles

In some embodiments, a virus-like particle (VLP) is provided thatincludes a disclosed immunogen (e.g., a recombinant HIV-1 Env ectodomainor immunogenic fragment thereof). VLPs lack the viral components thatare required for virus replication and thus represent a highlyattenuated form of a virus. The VLP can display a polypeptide (e.g., arecombinant HIV-1 Env protein) that is capable of eliciting an immuneresponse to HIV when administered to a subject. Virus like particles andmethods of their production are known and familiar to the person ofordinary skill in the art, and viral proteins from several viruses areknown to form VLPs, including human papillomavirus, HIV (Kang et al.,Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus (Notka et al.,Biol. Chem. 380: 341-52 (1999)), human polyomavirus (Goldmann et al., J.Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al., Vaccine 17: 1005-13(1999)), parvovirus (Casal, Biotechnology and Applied Biochemistry, Vol29, Part 2, pp 141-150 (1999)), canine parvovirus (Hurtado et al., J.Virol. 70: 5422-9 (1996)), hepatitis E virus (Li et al., J. Virol. 71:7207-13 (1997)), and Newcastle disease virus. The formation of such VLPscan be detected by any suitable technique. Examples of suitabletechniques known in the art for detection of VLPs in a medium include,e.g., electron microscopy techniques, dynamic light scattering (DLS),selective chromatographic separation (e.g., ion exchange, hydrophobicinteraction, and/or size exclusion chromatographic separation of theVLPs) and density gradient centrifugation.

The virus like particle can include any of the recombinant HIV-1 Envectodomain trimers or immunogenic fragments thereof, that are disclosedherein. For example, the virus like particle can include the recombinantHIV-1 Env ectodomain trimer or immunogenic fragments thereof, of any ofclaims X-Y included in the claim set below. Embodiments concerning thevirus-like particles are further described in Clauses 1-16, below.

Clause 1. A virus like particle comprising the recombinant HIV-1 Envectodomain trimer or immunogenic fragment thereof of any one of claims1-67;

particularly wherein the recombinant HIV-1 Env ectodomain trimer orimmunogenic fragment thereof is linked to a transmembrane domain;

particularly wherein the recombinant HIV-1 Env ectodomain trimercomprises DS and SOS substitutions as described herein;

particularly wherein the recombinant HIV-1 Env ectodomain trimer is achimeric HIV-1 Env trimer comprising a BG505 “platform” as describedherein, a V1V2 domain from a CAP256.SU (SEQ ID NO: 51), a BB201.B42 (SEQID NO: 81), a KER2018.11 (SEQ ID NO: 107), a CH070.1 (SEQ ID NO: 174), aZM233.6 (SEQ ID NO: 745), a Q23.17 (SEQ ID NO: 746), a A244 (SEQ ID NO:747), a T250-4 (SEQ ID NO: 2114), or a WITO.33 (SEQ ID NO: 748) strainof HIV-1, with the remainder of the HIV-1 Env ectodomain based on Envfrom a 45_01dG5 Env or a 426c Env that further comprises amino acidsubstitutions to remove the N-linked glycan sequons at positions 276,460, 463.

Clause 2. An isolated nucleic acid molecule encoding the virus likeparticle of clause 1.

Clause 3. The nucleic acid molecule of clause 2, wherein the nucleicacid molecule encodes a precursor protein of the gp120/gp41 protomers inthe recombinant HIV-1 Env ectodomain trimer.

Clause 4. The nucleic acid molecule of clause 2 or clause 3, operablylinked to a promoter.

Clause 5. A vector comprising the nucleic acid molecule of clause 4.

Clause 6. An isolated host cell comprising the vector of clause 5.

Clause 7. The virus-like particle of any one of the prior clauses,wherein administration of an effective amount of the virus like particleinduces a neutralizing immune response to HIV-1 Env in the subject.

Clause 8. An immunogenic composition comprising an effective amount ofthe virus like particle of any one of the prior clauses, and apharmaceutically acceptable carrier.

Clause 9. The immunogenic composition of clause 8, further comprising anadjuvant.

Clause 10. A method for generating an immune response to HumanImmunodeficiency Virus type 1 (HIV-1) gp120 in a subject, comprisingadministering to the subject an effective amount of the immunogeniccomposition of clause 9 or clause 10, thereby generating the immuneresponse.

Clause 11. A method for treating or preventing a Human ImmunodeficiencyVirus type 1 (HIV-1) infection in a subject, comprising administering tothe subject a therapeutically effective amount of the immunogeniccomposition of clause 9 or clause 10, thereby treating the subject orpreventing HIV-1 infection of the subject.

Clause 12. The method of clause 10 clause 11, comprising a prime-boostadministration of the immunogenic composition.

Clause 13. The method of any of clauses 10-12, wherein the subject is atrisk of or has an HIV-1 infection.

Clause 14. A kit comprising the virus like particle, nucleic acidmolecule, vector, or composition, of any of clauses 1-9, andinstructions for using the kit.

Clause 15. Use of the virus like particle, nucleic acid molecule,vector, or composition of any of clauses 1-9, to inhibit or preventHIV-1 infection in a subject.

Clause 16. Use of the virus like particle, nucleic acid molecule,vector, or composition of any of clauses 1-9, to induce an immuneresponse to HIV-1 Env in a subject.

G. Viral Vectors

The nucleic acid molecules encoding the disclosed immunogens (e.g., arecombinant HIV-1 Env ectodomain stabilized in a prefusion mature closedconformation or an immunogenic fragment thereof) can be included in aviral vector, for example for expression of the antigen in a host cell,or for immunization of a subject as disclosed herein. In someembodiments, the viral vectors are administered to a subject as part ofa prime-boost vaccination. In several embodiments, the viral vectors areincluded in a vaccine, such as a primer vaccine or a booster vaccine foruse in a prime-boost vaccination.

In several examples, the viral vector can be replication-competent. Forexample, the viral vector can have a mutation in the viral genome thatdoes not inhibit viral replication in host cells. The viral vector alsocan be conditionally replication-competent. In other examples, the viralvector is replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used toexpress the disclosed antigens, including polyoma, i.e., SV40 (Madzak etal., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur.Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412;Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. GeneTher., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

In several embodiments, the viral vector can include an adenoviralvector that expresses a disclosed recombinant HIV-1 Env ectodomain orimmunogenic fragment thereof. Adenovirus from various origins, subtypes,or mixture of subtypes can be used as the source of the viral genome forthe adenoviral vector. Non-human adenovirus (e.g., simian, chimpanzee,gorilla, avian, canine, ovine, or bovine adenoviruses) can be used togenerate the adenoviral vector. For example, a simian adenovirus can beused as the source of the viral genome of the adenoviral vector. Asimian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27,33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. Asimian adenovirus can be referred to by using any suitable abbreviationknown in the art, such as, for example, SV, SAdV, SAV or sAV. In someexamples, a simian adenoviral vector is a simian adenoviral vector ofserotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, achimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al.,Vaccine, 27:1293-1300, 2009). Human adenovirus can be used as the sourceof the viral genome for the adenoviral vector. Human adenovirus can beof various subgroups or serotypes. For instance, an adenovirus can be ofsubgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2,5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22,23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E(e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), anunclassified serogroup (e.g., serotypes 49 and 51), or any otheradenoviral serotype. The person of ordinary skill in the art is familiarwith replication competent and deficient adenoviral vectors (includingsingly and multiply replication deficient adenoviral vectors). Examplesof replication-deficient adenoviral vectors, including multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and7,195,896, and International Patent Application Nos. WO 94/28152, WO95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO97/21826, and WO 03/022311.

H. Neutralizing Immune Response

A disclosed recombinant HIV-1 Env ectodomain stabilized in a prefusionmature closed conformation, or immunogenic fragment thereof, can be usedto elicit a neutralizing immune response to HIV-1 in a subject. Inseveral such embodiments, induction of the immune response includesproduction of neutralizing antibodies to HIV-1.

In several embodiments, following immunization of a subject with adisclosed immunogen (e.g., as described herein) serum can be collectedfrom the subject at appropriate time points, frozen, and stored forneutralization testing. Methods to assay for neutralization activity areknown to the person of ordinary skill in the art and are furtherdescribed herein, and include, but are not limited to, plaque reductionneutralization (PRNT) assays, microneutralization assays, flow cytometrybased assays, single-cycle infection assays (e.g., as described inMartin et al (2003) Nature Biotechnology 21:71-76), and pseudovimsneutralization assays (e.g., as described in Georgiev et al. (Science,340, 751-756, 2013), Seaman et al. (J. Virol., 84, 1439-1452, 2005), andMascola et al. (J. Virol., 79, 10103-10107, 2005), each of which isincorporated by reference herein in its entirety.

In some embodiments, the serum neutralization activity can be assayedusing a panel of HIV-1 pseudovimses as described in Georgiev et al.,Science, 340, 751-756, 2013 or Seaman et al. J. Virol., 84, 1439-1452,2005. Briefly, pseudovirus stocks are prepared by co-transfection of293T cells with an HIV-1 Env-deficient backbone and an expressionplasmid encoding the Env gene of interest. The serum to be assayed isdiluted in Dulbecco's modified Eagle medium-10% FCS (Gibco) and mixedwith pseudovirus. After 30 min, 10,000 TZM-bl cells are added, and theplates are incubated for 48 hours. Assays are developed with aluciferase assay system (Promega, Madison, Wis.), and the relative lightunits (RLU) are read on a luminometer (Perkin-Elmer, Waltham, Mass.). Toaccount for background, a cutoff of ID₅₀≥40 can be used as a criterionfor the presence of serum neutralization activity against a givenpseudovirus.

In some embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogens to a subject (e.g., bya prime-boost administration of a DNA vector encoding a disclosedimmunogen (prime) followed by a protein nanoparticle including adisclosed immunogen (boost)) induces a neutralizing immune response inthe subject. In several embodiments, the neutralizing immune responsecan be detected using a pseudovirus neutralization assay against a panelof HIV-1 pseudoviruses including HIV-1 Env proteins from different HIV-1strains. In one example, the panel can include pseudoviruses includingEnv proteins from HIV-1 strains from Clade A (KER2018.11, Q23.17,Q168.a2, Q769.h5, and RW020.2), Clade B (BaL.01, 6101.10, BG1168.01,CAAN.A2, JR-FL, JR-CSF.JB, PVO.4, THR04156.18, TRJ04551.58, TRO.11, andYU2), and Clade C (DU156.12, DU422.01, ZAO12.29, ZM55.28a, and ZM106.9).In other examples, the panel can include pseudoviruses including Envproteins from the HIV-1 strains listed in Table S5 or Table S6 ofGeorgiev et al. (Science, 340, 751-756, 2013, which is incorporated byreference herein in its entirety), or Table 1 of Seaman et al. (J.Virol., 84, 1439-1452, 2005, which is incorporated by reference hereinin its entirety).

In some embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogen to a subject (e.g., bya prime-boost administration of a DNA vector encoding a disclosedimmunogen (prime) followed by a protein nanoparticle including adisclosed immunogen (boost)) induces a neutralizing immune response inthe subject, wherein serum from the subject neutralizes, with anID₅₀≥40, at least 30% (such as at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, or at least 90%) of pseudoviruses is a panelof pseudovimses including the HIV-1 Env proteins listed in Table S5 orTable S6 of Georgiev et al. (Science, 340, 751-756, 2013), or Table 1 ofSeaman et al. (J. Virol., 84, 1439-1452, 2005) or including Env proteinsfrom HIV-1 strains from Clade A (KER2018.11, Q23.17, Q168.a2, Q769.h5,and RW020.2), Clade B (BaL.01, 6101.10, BG1168.01, CAAN.A2, JR-FL,JR-CSF.JB, PVO.4, THR04156.18, TRJ04551.58, TRO.11, and YU2), and CladeC (DU156.12, DU422.01, ZA012.29, ZM55.28a, and ZM106.9).

In additional embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogen to a subject (e.g., bya prime-boost administration of a DNA vector encoding a disclosedimmunogen (prime) followed by a protein nanoparticle including adisclosed immunogen (boost)) induces a neutralizing immune response inthe subject, wherein serum from the subject neutralizes, with anID₅₀≥40, at least 30% (such as at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, or at least 90%) of pseudoviruses is a panelof pseudoviruses including the Clade A, Clade B, or Clade C HIV-1 Envproteins listed in Table S5 or Table S6 of Georgiev et al. (Science,340, 751-756, 2013), or Table 1 of Seaman et al. (J. Virol., 84,1439-1452, 2005) or including Env proteins from HIV-1 strains from CladeA (KER2018.11, Q23.17, Q168.a2, Q769.h5, and RW020.2), Clade B (BaL.01,6101.10, BG1168.01, CAAN.A2, JR-FL, JR-CSF.JB, PVO.4, THR04156.18,TRJO4551.58, TRO.11, and YU2), and Clade C (DU156.12, DU422.01,ZA012.29, ZM55.28a, and ZM106.9).

I. Compositions

The disclosed immunogens (for example, a recombinant HIV-1 Envectodomain or immunogenic fragment thereof, or a protein nanoparticleincluding such proteins), or nucleic acid molecule encoding animmunogen, can be included in a pharmaceutical composition (includingtherapeutic and prophylactic formulations), often combined together withone or more pharmaceutically acceptable vehicles and, optionally, othertherapeutic ingredients (for example, antibiotics or antiviral drugs).In several embodiments, pharmaceutical compositions including one ormore of the disclosed immunogens are immunogenic compositions.

Such pharmaceutical compositions can be administered to subjects by avariety of administration modes known to the person of ordinary skill inthe art, for example, intramuscular, subcutaneous, intravenous,intra-arterial, intra-articular, intraperitoneal, or parenteral routes.

To formulate the pharmaceutical compositions, the disclosed immunogens(for example, a recombinant HIV-1 Env ectodomain or immunogenic fragmentthereof, or a protein nanoparticle including such proteins), or nucleicacid molecule encoding an immunogen, can be combined with variouspharmaceutically acceptable additives, as well as a base or vehicle fordispersion of the conjugate. Desired additives include, but are notlimited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, and the like. In addition,local anesthetics (for example, benzyl alcohol), isotonizing agents (forexample, sodium chloride, mannitol, sorbitol), adsorption inhibitors(for example, TWEEN® 80), solubility enhancing agents (for example,cyclodextrins and derivatives thereof), stabilizers (for example, serumalbumin), and reducing agents (for example, glutathione) can beincluded. Adjuvants, such as aluminum hydroxide (ALHYDROGEL®, availablefrom Brenntag Biosector, Copenhagen, Denmark and AMPHOGEL®, WyethLaboratories, Madison, N.J.), Freund's adjuvant, MPL™ (3-O-deacylatedmonophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (GeneticsInstitute, Cambridge, Mass.), among many other suitable adjuvants wellknown in the art, can be included in the compositions.

When the composition is a liquid, the tonicity of the formulation, asmeasured with reference to the tonicity of 0.9% (w/v) physiologicalsaline solution taken as unity, is typically adjusted to a value atwhich no substantial, irreversible tissue damage will be induced at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about 0.3 to about 3.0, such as about 0.5 toabout 2.0, or about 0.8 to about 1.7.

The disclosed immunogens (for example, a recombinant HIV-1 Envectodomain or immunogenic fragment thereof, or a protein nanoparticleincluding such proteins), or nucleic acid molecule encoding an immunogencan be dispersed in a base or vehicle, which can include a hydrophiliccompound having a capacity to disperse the antigens, and any desiredadditives. The base can be selected from a wide range of suitablecompounds, including but not limited to, copolymers of polycarboxylicacids or salts thereof, carboxylic anhydrides (for example, maleicanhydride) with other monomers (for example, methyl (meth)acrylate,acrylic acid and the like), hydrophilic vinyl polymers, such aspolyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulosederivatives, such as hydroxymethylcellulose, hydroxypropylcellulose andthe like, and natural polymers, such as chitosan, collagen, sodiumalginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof.Often, a biodegradable polymer is selected as a base or vehicle, forexample, polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters and the like can be employed as vehicles. Hydrophilicpolymers and other vehicles can be used alone or in combination, andenhanced structural integrity can be imparted to the vehicle by partialcrystallization, ionic bonding, cross-linking and the like. The vehiclecan be provided in a variety of forms, including fluid or viscoussolutions, gels, pastes, powders, microspheres and films, for examplesfor direct application to a mucosal surface.

The disclosed immunogens (for example, a recombinant HIV-1 Envectodomain or immunogenic fragment thereof, or a protein nanoparticleincluding such proteins), or nucleic acid molecule encoding an immunogencan be combined with the base or vehicle according to a variety ofmethods, and release of the antigens can be by diffusion, disintegrationof the vehicle, or associated formation of water channels. In somecircumstances, the disclosed immunogens (for example, a recombinantHIV-1 Env ectodomain or immunogenic fragment thereof, or a proteinnanoparticle including such proteins), or nucleic acid molecule encodingan immunogen is dispersed in microcapsules (microspheres) ornanocapsules (nanospheres) prepared from a suitable polymer, forexample, isobutyl 2-cyanoacrylate (see, for example, Michael et al., J.Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatibledispersing medium, which yields sustained delivery and biologicalactivity over a protracted time.

The pharmaceutical compositions of the disclosure can alternativelycontain as pharmaceutically acceptable vehicles substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate, andtriethanolamine oleate. For solid compositions, conventional nontoxicpharmaceutically acceptable vehicles can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

Pharmaceutical compositions for administering the immunogeniccompositions can also be formulated as a solution, microemulsion, orother ordered structure suitable for high concentration of activeingredients. The vehicle can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), andsuitable mixtures thereof. Proper fluidity for solutions can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of a desired particle size in the case of dispersibleformulations, and by the use of surfactants. In many cases, it will bedesirable to include isotonic agents, for example, sugars, polyalcohols,such as mannitol and sorbitol, or sodium chloride in the composition.Prolonged absorption of the disclosed antigens can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

In certain embodiments, the disclosed immunogens (for example, arecombinant HIV-1 Env ectodomain or immunogenic fragment thereof, or aprotein nanoparticle including such proteins), or nucleic acid moleculeencoding an immunogen can be administered in a time-release formulation,for example in a composition that includes a slow release polymer. Thesecompositions can be prepared with vehicles that will protect againstrapid release, for example a controlled release vehicle such as apolymer, microencapsulated delivery system or bioadhesive gel. Prolongeddelivery in various compositions of the disclosure can be brought aboutby including in the composition agents that delay absorption, forexample, aluminum monostearate hydrogels and gelatin. When controlledrelease formulations are desired, controlled release binders suitablefor use in accordance with the disclosure include any biocompatiblecontrolled release material which is inert to the active agent and whichis capable of incorporating the disclosed antigen and/or otherbiologically active agent. Numerous such materials are known in the art.Useful controlled-release binders are materials that are metabolizedslowly under physiological conditions following their delivery (forexample, at a mucosal surface, or in the presence of bodily fluids).Appropriate binders include, but are not limited to, biocompatiblepolymers and copolymers well known in the art for use in sustainedrelease formulations. Such biocompatible compounds are non-toxic andinert to surrounding tissues, and do not trigger significant adverseside effects, such as nasal irritation, immune response, inflammation,or the like. They are metabolized into metabolic products that are alsobiocompatible and easily eliminated from the body. Numerous systems forcontrolled delivery of therapeutic proteins are known (e.g., U.S. Pat.Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735;and 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the conjugate in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thedisclosed antigen and/or other biologically active agent into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the disclosed antigen plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The prevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

In several embodiments, the compositions include an adjuvant. The personof ordinary skill in the art is familiar with adjuvants, for example,those that can be included in an immunogenic composition. It will beappreciated that the choice of adjuvant can be different in thesedifferent applications, and the optimal adjuvant and concentration foreach situation can be determined empirically by those of skill in theart.

The pharmaceutical composition typically contains a therapeuticallyeffective amount of a disclosed immunogen (for example, a recombinantHIV-1 Env ectodomain or immunogenic fragment thereof, or a proteinnanoparticle including such proteins), or nucleic acid molecule encodingan immunogen, or viral vector can be prepared by conventionaltechniques. Preparation of immunogenic compositions, including those foradministration to human subjects, is generally described inPharmaceutical Biotechnology, Vol. 61 Vaccine Design-the subunit andadjuvant approach, edited by Powell and Newman, Plenum Press, 1995. NewTrends and Developments in Vaccines, edited by Voller et al., UniversityPark Press, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomesis described, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757. Typically, the amount of antigen in each dose of theimmunogenic composition is selected as an amount which induces an immuneresponse without significant, adverse side effects.

The amount of the disclosed immunogen (for example, a recombinant HIV-1Env ectodomain or immunogenic fragment thereof, or a proteinnanoparticle including such proteins), or nucleic acid molecule encodingan immunogen, or viral vector can vary depending upon the specificantigen employed, the route and protocol of administration, and thetarget population, for example. For protein therapeutics, typically,each human dose will comprise 1-1000 μg of protein, such as from about 1μg to about 100 μg, for example, from about 1 μg to about 50 μg, such asabout 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg. The amountutilized in an immunogenic composition is selected based on the subjectpopulation (e.g., infant or elderly). An optimal amount for a particularcomposition can be ascertained by standard studies involving observationof antibody titers and other responses in subjects. It is understoodthat a therapeutically effective amount of a disclosed immunogen, suchas a recombinant HIV-1 Env ectodomain or fragment thereof, proteinnanoparticle, viral vector, or nucleic acid molecule in a immunogeniccomposition, can include an amount that is ineffective at eliciting animmune response by administration of a single dose, but that iseffective upon administration of multiple dosages, for example in aprime-boost administration protocol.

In some embodiments, the composition can be provided as a sterilecomposition. In more embodiments, the composition can be provided inunit dosage form for use to induce an immune response in a subject, forexample, to prevent HIV-1 infection in the subject. A unit dosage formcontains a suitable single preselected dosage for administration to asubject, or suitable marked or measured multiples of two or morepreselected unit dosages, and/or a metering mechanism for administeringthe unit dose or multiples thereof. In other embodiments, thecomposition further includes an adjuvant.

J. Therapeutic Methods

The recombinant HIV-1 Env proteins, immunogenic fragments thereof,protein nanoparticles, polynucleotides encoding the recombinant HIV-1Env proteins or immunogenic fragments, vectors and compositions, can beused in methods of preventing, inhibiting and treating an HIV-1infection, as well as methods of inducing an immune response to HIV-1,as described below. In several embodiments, a therapeutically effectiveamount of an immunogenic composition including one or more of thedisclosed recombinant HIV-1 Env proteins or immunogenic fragmentsthereof, or protein nanoparticles of VLPs, or nucleic acid molecule orviral vector encoding a recombinant HIV-1 Env proteins or immunogenicfragments thereof, can be administered to a subject in order to generatean immune response to HIV-1.

In some embodiments, a subject is selected for treatment that has, or isat risk for developing, an HIV infection, for example because ofexposure or the possibility of exposure to HIV. Following administrationof a therapeutically effective amount of a recombinant HIV-1 Envproteins, immunogenic fragments thereof, protein nanoparticles,polynucleotides encoding the recombinant HIV-1 Env proteins orimmunogenic fragments, vectors and compositions, the subject can bemonitored for HIV-1 infection, symptoms associated with HIV-1 infection,or both.

Typical subjects intended for treatment with the therapeutics andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease or condition, or to determine the statusof an existing disease or condition in a subject. These screeningmethods include, for example, conventional work-ups to determineenvironmental, familial, occupational, and other such risk factors thatmay be associated with the targeted or suspected disease or condition,as well as diagnostic methods, such as various ELISA and otherimmunoassay methods, which are available and well known in the art todetect and/or characterize HIV infection. These and other routinemethods allow the clinician to select patients in need of therapy usingthe methods and pharmaceutical compositions of the disclosure. Inaccordance with these methods and principles, a composition can beadministered according to the teachings herein, or other conventionalmethods known to the person of ordinary skill in the art, as anindependent prophylaxis or treatment program, or as a follow-up, adjunctor coordinate treatment regimen to other treatments.

The methods can be used to inhibit, treat or prevent HIV infection invivo. When inhibiting, treating, or preventing infection in vivo, themethods can be used either to avoid infection in an HIV-seronegativesubject (e.g., by inducing an immune response that protects againstHIV-1 infection), or to treat existing infection in an HIV-seropositivesubject. The HIV-seropositive subject may or may not carry a diagnosisof AIDS. Hence in some embodiments the methods involves selecting asubject at risk for contracting HIV infection, or a subject at risk ofdeveloping AIDS (such as a subject with HIV infection), andadministering a recombinant HIV-1 Env proteins, immunogenic fragmentsthereof, protein nanoparticles, polynucleotides encoding the recombinantHIV-1 Env proteins or immunogenic fragments, vectors and compositions,to the subject.

Treatment of HIV by inhibiting HIV replication or infection can includedelaying the development of AIDS in a subject. Treatment of HIV can alsoinclude reducing signs or symptoms associated with the presence of HIV(for example by reducing or inhibiting HIV replication). In someexamples, treatment using the methods disclosed herein prolongs the timeof survival of the subject.

The administration of a disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition can be for prophylactic or therapeutic purpose.When provided prophylactically, the disclosed therapeutic agents areprovided in advance of any symptom, for example in advance of infection.The prophylactic administration of the disclosed therapeutic agentsserves to prevent or ameliorate any subsequent infection. When providedtherapeutically, the disclosed therapeutic agents are provided at orafter the onset of a symptom of disease or infection, for example afterdevelopment of a symptom of HIV-1 infection, or after diagnosis of HIV-1infection. The therapeutic agents can thus be provided prior to theanticipated exposure to HIV virus so as to attenuate the anticipatedseverity, duration or extent of an infection and/or associated diseasesymptoms, after exposure or suspected exposure to the virus, or afterthe actual initiation of an infection.

The immunogenic composition including one or more of the disclosedagents (for example, a recombinant HIV-1 Env protein, immunogenicfragments thereof, protein nanoparticles, or VLP), or nucleic acidmolecule or viral vector encoding a recombinant HIV-1 Env ectodomain orimmunogenic fragment thereof, can be used in coordinate vaccinationprotocols or combinatorial formulations. In certain embodiments, novelcombinatorial immunogenic compositions and coordinate immunizationprotocols employ separate immunogens or formulations, each directedtoward eliciting an anti-HIV immune response, such as an immune responseto HIV-1 Env protein. Separate immunogenic compositions that elicit theanti-HIV immune response can be combined in a polyvalent immunogeniccomposition administered to a subject in a single immunization step, orthey can be administered separately (in monovalent immunogeniccompositions) in a coordinate immunization protocol.

HIV infection does not need to be completely eliminated or reduced orprevented for the methods to be effective. For example, treatment withone or more of the disclosed therapeutic agents can reduce or inhibitHIV infection by a desired amount, for example by at least 10%, at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100% (elimination orprevention of detectable HIV infected cells), as compared to HIVinfection in the absence of the therapeutic agent. In additionalexamples, HIV replication can be reduced or inhibited by the disclosedmethods. HIV replication does not need to be completely eliminated forthe method to be effective. For example, treatment with one or more ofthe disclosed recombinant HIV-1 Env proteins, immunogenic fragmentthereof, protein nanoparticles, polynucleotides encoding a recombinantHIV-1 Env ectodomain or immunogenic fragment, vectors or compositionscan HIV replication by a desired amount, for example by at least 10%, atleast 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%(elimination or prevention of detectable HIV replication), as comparedto HIV replication in the absence of the therapeutic agent.

To successfully reproduce itself, HIV must convert its RNA genome toDNA, which is then imported into the host cell's nucleus and insertedinto the host genome through the action of HIV integrase. Because HIV'sprimary cellular target, CD4+ T-Cells, can function as the memory cellsof the immune system, integrated HIV can remain dormant for the durationof these cells' lifetime. Memory T-Cells may survive for many years andpossibly for decades. This latent HIV reservoir can be measured byco-culturing CD4+ T-Cells from infected patients with CD4+ T-Cells fromuninfected donors and measuring HIV protein or RNA (See, e.g., Archin etal., AIDS, 22:1131-1135, 2008). In some embodiments, the providedmethods of treating or inhibiting HIV infection include reduction orelimination of the latent reservoir of HIV infected cells in a subject.For example, a reduction of at least 10%, at least 20%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or even at least 100% (elimination of detectable HIV) of thelatent reservoir of HIV infected cells in a subject, as compared to thelatent reservoir of HIV infected cells in a subject in the absence ofthe treatment with one or more of the provided recombinant HIV-1 Envproteins, immunogenic fragments thereof, protein nanoparticles,polynucleotides encoding a recombinant HIV-1 Env ectodomain orimmunogenic fragment, vectors or compositions.

Studies have shown that the rate of HIV transmission from mother toinfant is reduced significantly when zidovudine is administered toHIV-infected women during pregnancy and delivery and to the offspringafter birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541).Several studies of mother-to-infant transmission of HIV havedemonstrated a correlation between the maternal virus load at deliveryand risk of HIV transmission to the child. The disclosed recombinantHIV-1 Env proteins, immunogenic fragments thereof, proteinnanoparticles, polynucleotides encoding a recombinant HIV-1 Envectodomain or immunogenic fragment, vectors and compositions are of usein decreasing HIV-transmission from mother to infant. Thus, in someembodiments a therapeutically effective amount of one or more of theprovided therapeutic agents is administered in order to preventtransmission of HIV, or decrease the risk of transmission of HIV, from amother to an infant. In some embodiments, a therapeutically effectiveamount of the agent can be administered to a pregnant subject to inducean immune response that generates neutralizing antibodies that arepasses to the fetus via the umbilical cord to protect the fetus frominfection during birth. In some embodiments, both a therapeuticallyeffective amount of a disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition and a therapeutically effective amount of anotheranti-HIV agent, such as zidovudine, is administered to the mother and/orinfant.

Administration of a therapeutically effective amount of a disclosedrecombinant HIV-1 Env protein, immunogenic fragment thereof, proteinnanoparticle, polynucleotide encoding a recombinant HIV-1 Env ectodomainor immunogenic fragment, vector or composition induces a sufficientimmune response to treat or inhibit or prevent the pathogenic infection,for example, to inhibit the infection and/or reduce the signs and/orsymptoms of the infection. Amounts effective for this use will dependupon the severity of the disease, the general state of the subject'shealth, and the robustness of the subject's immune system.

For prophylactic and therapeutic purposes, a therapeutically effectiveamount of a disclosed recombinant HIV-1 Env protein, immunogenicfragment thereof, protein nanoparticle, polynucleotide encoding arecombinant HIV-1 Env ectodomain or immunogenic fragment, vector orcomposition can be administered to the subject in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the therapeutic agents can be provided as repeated doseswithin a prolonged prophylaxis or treatment regimen that will yieldclinically significant results to alleviate one or more symptoms ordetectable conditions associated with a targeted disease or condition asset forth herein.

In several embodiments, a prime-boost immunization protocol is used, anda recombinant HIV-1 Env ectodomain trimer including that binds to matureand unmutated common ancestor (UCA) forms of multiple classes of broadlyneutralizing antibodies (e.g., targeting the CD4 binding site and theV1V2 domain) is used for the prime, and (in some embodiments, also forthe boost. Exemplary recombinant HIV-1 Env ectodomain fur use as a primein such embodiments are provided herein and include those set forth asSEQ ID NOs: SEQ ID NOs: 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153,2154, 2155, 2156, 2157, 2158, and 2159.

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject, orthat induce a desired response in the subject (such as a neutralizingimmune response). Suitable models in this regard include, for example,murine, rat, porcine, feline, ferret, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using in vitro models (for example,immunologic and histopathologic assays). Using such models, onlyordinary calculations and adjustments are required to determine anappropriate concentration and dose to administer a therapeuticallyeffective amount of the composition (for example, amounts that areeffective to elicit a desired immune response or alleviate one or moresymptoms of a targeted disease). In alternative embodiments, aneffective amount or effective dose of the composition may simply inhibitor enhance one or more selected biological activities correlated with adisease or condition, as set forth herein, for either therapeutic ordiagnostic purposes.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, systemic circulation).Higher or lower concentrations can be selected based on the mode ofdelivery, for example, trans-epidermal, rectal, oral, pulmonary, orintranasal delivery versus intravenous or subcutaneous delivery. Theactual dosage of disclosed recombinant HIV-1 Env ectodomain, immunogenicfragment thereof, protein nanoparticle, polynucleotide encoding arecombinant HIV-1 Env ectodomain or immunogenic fragment, vector orcomposition will vary according to factors such as the diseaseindication and particular status of the subject (for example, thesubject's age, size, fitness, extent of symptoms, susceptibilityfactors, and the like), time and route of administration, other drugs ortreatments being administered concurrently, as well as the specificpharmacology of the composition for eliciting the desired activity orbiological response in the subject. Dosage regimens can be adjusted toprovide an optimum prophylactic or therapeutic response. As describedabove in the forgoing listing of terms, a therapeutically effectiveamount is also one in which any toxic or detrimental side effects of thedisclosed immunogen and/or other biologically active agent is outweighedin clinical terms by therapeutically beneficial effects.

A non-limiting range for a therapeutically effective amount of thedisclosed immunogen (e.g., a recombinant HIV-1 Env protein, or nucleicacid encoding such protein, or nanoparticle including such protein)within the methods and immunogenic compositions of the disclosure isabout 0.0001 mg/kg body weight to about 10 mg/kg body weight, such asabout 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg,about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg,about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg,about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg,about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg,or about 10 mg/kg, for example 0.01 mg/kg to about 1 mg/kg body weight,about 0.05 mg/kg to about 5 mg/kg body weight, about 0.2 mg/kg to about2 mg/kg body weight, or about 1.0 mg/kg to about 10 mg/kg body weight.

In some embodiments, the dosage includes a set amount of a disclosedimmunogen (e.g., a recombinant HIV-1 Env protein, or nucleic acidencoding such protein, or nanoparticle including such protein) such asfrom about 1-300 μg, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, orabout 300 μg. The dosage and number of doses will depend on the setting,for example, in an adult or anyone primed by prior HIV infection orimmunization, a single dose may be a sufficient booster. In naïvesubjects, in some examples, at least two doses would be given, forexample, at least three doses. In some embodiments, an annual boost isgiven, for example, along with an annual influenza vaccination.

Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

In several embodiments, it may be advantageous to administer thetherapeutic agents disclosed herein with other agents such as proteins,peptides, antibodies, and other antiviral agents, such as anti-HIVagents. Examples of such anti-HIV therapeutic agents include nucleosidereverse transcriptase inhibitors, such as abacavir, AZT, didanosine,emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine,zidovudine, and the like, non-nucleoside reverse transcriptaseinhibitors, such as delavirdine, efavirenz, nevirapine, proteaseinhibitors such as amprenavir, atazanavir, indinavir, lopinavir,nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir, and thelike, and fusion protein inhibitors such as enfuvirtide and the like. Insome examples, the disclosed therapeutic agents are administered withT-helper cells, such as exogenous T-helper cells. Exemplary methods forproducing and administering T-helper cells can be found in InternationalPatent Publication WO 03/020904, which is incorporated herein byreference.

For any application, treatment with a disclosed recombinant HIV-1 Envprotein, immunogenic fragment thereof, protein nanoparticle,polynucleotide encoding a recombinant HIV-1 Env ectodomain orimmunogenic fragment, vector or composition can be combined withanti-retroviral therapy, such as HAART. Antiretroviral drugs are broadlyclassified by the phase of the retrovims life-cycle that the druginhibits. The therapeutic agents can be administered before, during,concurrent to and/or after retroviral therapy. In some embodiments, thetherapeutic agents are administered following a course of retroviraltherapy. The disclosed therapeutic agents can be administered inconjunction with nucleoside and nucleotide reverse transcriptaseinhibitors (nRTI), non-nucleoside reverse transcriptase inhibitors(NNRTI), protease inhibitors, Entry inhibitors (or fusion inhibitors),Maturation inhibitors, or a broad spectrum inhibitors, such as naturalantivirals. Exemplary agents include lopinavir, ritonavir, zidovudine,lamivudine, tenofovir, emtricitabine and efavirenz.

In some embodiments, a disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition can be used as an immunogen to prime or induce animmune response (such as a T or B cell response) to HIV-1 in a subject.In some such embodiments, the T cell response is a CD4⁺ T helper cellresponse, such as a Th1 cell response.

In some embodiments, the disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition is administered to the subject simultaneously withthe administration of the adjuvant. In other embodiments, therecombinant HIV-1 Env protein, immunogenic fragment thereof, proteinnanoparticle, polynucleotide encoding a recombinant HIV-1 Env ectodomainor immunogenic fragment, vector or composition is administered to thesubject after the administration of the adjuvant and within a sufficientamount of time to induce the immune response.

The recombinant HIV-1 Env protein, immunogenic fragment thereof, proteinnanoparticle, polynucleotide encoding a recombinant HIV-1 Env ectodomainor immunogenic fragment, vector or composition can be used in coordinatevaccination protocols or combinatorial formulations. In certainembodiments, combinatorial and coordinate immunization protocols employseparate immunogens or formulations, each directed toward eliciting ananti-HIV immune response, such as an immune response to HIV-1 Env.Separate immunogenic compositions that elicit the anti-HIV immuneresponse can be combined in a polyvalent immunogenic compositionadministered to a subject in a single immunization step, or they can beadministered separately (in monovalent immunogenic compositions) in acoordinate immunization protocol.

In one embodiment, a suitable immunization regimen includes at least twoseparate inoculations with one or more immunogenic compositions, with asecond inoculation being administered more than about two, about threeto eight, or about four, weeks following the first inoculation. A thirdinoculation can be administered several months after the secondinoculation, and in specific embodiments, more than about five monthsafter the first inoculation, more than about six months to about twoyears after the first inoculation, or about eight months to about oneyear after the first inoculation. Periodic inoculations beyond the thirdare also desirable to enhance the subject's “immune memory.” Theadequacy of the vaccination parameters chosen, e.g., formulation, dose,regimen and the like, can be determined by taking aliquots of serum fromthe subject and assaying antibody titers during the course of theimmunization program. Alternatively, the T cell populations can bemonitored by conventional methods. In addition, the clinical conditionof the subject can be monitored for the desired effect, e.g., preventionof HIV-1 infection or progression to AIDS, improvement in disease state(e.g., reduction in viral load), or reduction in transmission frequencyto an uninfected partner. If such monitoring indicates that vaccinationis sub-optimal, the subject can be boosted with an additional dose ofimmunogenic composition, and the vaccination parameters can be modifiedin a fashion expected to potentiate the immune response. Thus, forexample, the dose of the disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition and/or adjuvant can be increased or the route ofadministration can be changed.

It is contemplated that there can be several boosts, and that each boostcan be a different disclosed recombinant HIV-1 Env protein, immunogenicfragment thereof, protein nanoparticle, polynucleotide encoding arecombinant HIV-1 Env ectodomain or immunogenic fragment, vector orcomposition. It is also contemplated in some examples that the boost maybe the same recombinant HIV-1 Env protein, immunogenic fragment thereof,protein nanoparticle, polynucleotide encoding a recombinant HIV-1 Envectodomain or immunogenic fragment, vector or composition as anotherboost, or the prime.

The prime can be administered as a single dose or multiple doses, forexample two doses, three doses, four doses, five doses, six doses ormore can be administered to a subject over days, weeks or months. Theboost can be administered as a single dose or multiple doses, forexample two to six doses, or more can be administered to a subject overa day, a week or months. Multiple boosts can also be given, such one tofive, or more. Different dosages can be used in a series of sequentialinoculations. For example a relatively large dose in a primaryinoculation and then a boost with relatively smaller doses. The immuneresponse against the selected antigenic surface can be generated by oneor more inoculations of a subject.

Upon administration of a disclosed recombinant HIV-1 Env protein,immunogenic fragment thereof, protein nanoparticle, polynucleotideencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vector or composition of this disclosure, the immune system of thesubject typically responds to the immunogenic composition by producingantibodies specific for HIV-1 Env protein. Such a response signifiesthat an immunologically effective dose was delivered to the subject.

An immunologically effective dosage can be achieved by single ormultiple administrations (including, for example, multipleadministrations per day), daily, or weekly administrations. For eachparticular subject, specific dosage regimens can be evaluated andadjusted over time according to the individual need and professionaljudgment of the person administering or supervising the administrationof the immunogenic composition. In some embodiments, the antibodyresponse of a subject will be determined in the context of evaluatingeffective dosages/immunization protocols. In most instances it will besufficient to assess the antibody titer in serum or plasma obtained fromthe subject. Decisions as to whether to administer booster inoculationsand/or to change the amount of the therapeutic agent administered to theindividual can be at least partially based on the antibody titer level.The antibody titer level can be based on, for example, an immunobindingassay which measures the concentration of antibodies in the serum whichbind to an antigen including, for example, a disclosed recombinant HIV-1Env protein. The methods of using immunogenic composition, and therelated compositions and methods of the disclosure are useful inincreasing resistance to, preventing, ameliorating, and/or treatinginfection and disease caused by HIV (such as HIV-1) in animal hosts, andother, in vitro applications.

In certain embodiments, the recombinant HIV-1 Env protein, immunogenicfragment thereof, protein nanoparticle, polynucleotide encoding arecombinant HIV-1 Env ectodomain or immunogenic fragment, vector orcomposition is administered sequentially with other anti-HIV therapeuticagents, such as before or after the other agent. One of ordinary skillin the art would know that sequential administration can meanimmediately following or after an appropriate period of time, such ashours, days, weeks, months, or even years later.

In additional embodiments, a therapeutically effective amount of apharmaceutical composition including a nucleic acid encoding a disclosedrecombinant HIV-1 Env ectodomain or immunogenic fragment thereof isadministered to a subject, for example to generate an immune response.In one specific, non-limiting example, a therapeutically effectiveamount of a nucleic acid encoding a disclosed recombinant HIV-1 Envectodomain or immunogenic fragment thereof, is administered to a subjectto treat or prevent or inhibit HIV infection, or to induce an immuneresponse to HIV-1 (such as to gp120) in the subject.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a disclosedrecombinant HIV-1 Env ectodomain or immunogenic fragment thereof can beplaced under the control of a promoter to increase expression of themolecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a disclosedrecombinant HIV-1 Env ectodomain or immunogenic fragment thereof, canalso be expressed by attenuated viral hosts or vectors or bacterialvectors. Recombinant vaccinia virus, adeno-associated virus (AAV),herpes virus, retrovirus, cytogmeglo virus or other viral vectors can beused to express the peptide or protein, thereby eliciting a CTLresponse. For example, vaccinia vectors and methods useful inimmunization protocols are described in U.S. Pat. No. 4,722,848. BCG(Bacillus Calmette Guerin) provides another vector for expression of thepeptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed recombinant HIV-1Env ectodomain or immunogenic fragment thereof, is introduced directlyinto cells. For example, the nucleic acid can be loaded onto goldmicrospheres by standard methods and introduced into the skin by adevice such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acids can be“naked,” consisting of plasmids under control of a strong promoter.Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites, including tissues in proximity tometastases. Dosages for injection are usually around 0.5 μg/kg to about50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see,e.g., U.S. Pat. No. 5,589,466).

K. Immunodiagnostic Methods

In addition to the therapeutic methods provided above, any of thedisclosed immunogens (for example, disclosed recombinant HIV-1 Envectodomain or immunogenic fragment thereof) can be utilized to produceantigen specific immunodiagnostic reagents, for example, forserosurveillance. Immunodiagnostic reagents can be designed from any ofthe antigenic polypeptide described herein. For example, in the case ofthe disclosed immunogens, the presence of serum antibodies to HIV ismonitored using the isolated immunogens disclosed herein, such as todetect an HIV infection and/or the presence of antibodies thatspecifically bind to HIV-1 Env in a unliganded conformation.

Methods are further provided for a diagnostic assay to monitor HIV-1induced disease in a subject and/or to monitor the response of thesubject to immunization with one or more of the disclosed antigens. By“HIV-1 induced disease” is intended any disease caused, directly orindirectly, by HIV. An example of an HIV-1 induced disease is acquiredimmunodeficiency syndrome (AIDS). The method includes contacting adisclosed immunogen with a sample of bodily fluid from the subject, anddetecting binding of antibodies in the sample to the disclosedimmunogens. In addition, the detection of the HWV-1 binding antibodyalso allows the response of the subject to immunization with thedisclosed antigen to be monitored. In still other embodiments, the titerof the HIV-1 binding antibodies is determined. The binding can bedetected by any means known to one of skill in the art, including theuse of labeled secondary antibodies that specifically bind theantibodies from the sample. Labels include radiolabels, enzymaticlabels, and fluorescent labels. In other embodiments, a disclosedimmunogen is used to isolate antibodies present in a subject orbiological sample obtained from a subject.

Generally, the method includes contacting a sample from a subject, suchas, but not limited to a blood, serum, plasma, urine or sputum samplefrom the subject with one or more of the disclosed recombinant HWV-1 Envproteins or immunogenic fragments thereof (including a polymeric formthereof) and detecting binding of antibodies in the sample to thedisclosed immunogens. The binding can be detected by any means known toone of skill in the art, including the use of labeled secondaryantibodies that specifically bind the antibodies from the sample. Labelsinclude radiolabels, enzymatic labels, and fluorescent labels.

L. Kits

Any immunodiagnostic or therapeutic reagents can be provided ascomponents of a kit. Optionally, such a kit includes additionalcomponents including packaging, instructions and various other reagents,such as buffers, substrates, antibodies or ligands, such as controlantibodies or ligands, and detection reagents. The kit can include acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container typically holds a compositionincluding one or more of the disclosed recombinant HWV-1 Env proteins,immunogenic fragments thereof, protein nanoparticles, polynucleotidesencoding a recombinant HIV-1 Env ectodomain or immunogenic fragment,vectors or compositions, which is effective for treating, preventing,diagnosing, monitoring HIV infection or immune response. In severalembodiments the container may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The label orpackage insert indicates that the composition is used for treating theparticular condition.

The label or package insert typically will further include instructionsfor use of an antigen, or a nucleic acid or a viral vector encoding,expressing or including the antigen, for example, in a method oftreating or preventing a HIV infection. The package insert typicallyincludes instructions customarily included in commercial packages oftherapeutic products that contain information about the indications,usage, dosage, administration, contraindications and/or warningsconcerning the use of such therapeutic products. The instructionalmaterials may be written, in an electronic form (such as a computerdiskette or compact disk) or may be visual (such as video files). Thekits may also include additional components to facilitate the particularapplication for which the kit is designed. The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Structure, Activation, and Immune Recognition of PrefusionHIV-1 Env

This example illustrates the structure, activation, and immunerecognition of prefusion HIV-1 Env. The HIV-1-Env ectodomain trimer,comprising three gp120 and three gp41 subunits, is a conformationalmachine that facilitates HIV-1 entry by rearranging from a matureunliganded state, through receptor-bound intermediates, to a postfusionstate. This example shows the structure at 3.5-Å resolution for anHIV-1-Env trimer bound by antibodies PGT122 and 35O22. This structurereveals the prefusion conformation of gp41, indicates rearrangementsneeded for fusion activation, and defines parameters of immune evasionfor the antigenic target of most neutralizing antibodies. Prefusion gp41encircles extended N- and C-terminal strands of gp120 with a 4-helixcollar, which is fastened by insertion of a fusion peptide-proximalmethionine into a gp41-tryptophan clasp. Spike rearrangements requiredfor entry likely involve opening the clasp and expelling the termini.N-linked glycosylation and sequence-variable regions cover the matureectodomain trimer: the prevalence and location of effective neutralizingresponses from seroconverter and chronic cohorts are mapped, andalterations that stabilize its conformation are identified.

Initially synthesized as a gp160 precursor, which is cleaved into gp120and gp41 subunits, the trimeric HIV-1-Env ectodomain trimer displaysunusual posttranslational processing including the addition of 25-30N-linked glycans per gp120-gp41 protomer, tyrosine sulfation, and slowsignal peptide cleavage. It rearranges from a mature unliganded statethat evades antibody recognition, through intermediate states that bindto receptors CD4 and co-receptor (either CCR5 or CXCR4), to a postfusionstate (reviewed in Wyatt, R. & Sodroski, Science 280, 1884-1888, 1998).Over the last 20 years substantial atomic-level detail has been obtainedon these states, including structures of receptor-bound gp120 (Kwong etal. Nature 393, 648-659, 1998), postfusion gp41 (Chan at al., Cell 89,263-273, 1997; Weissenhom et al., Nature 387, 426-430, 1997), and thetrimeric arrangement of prefusion gp120 along with two gp41 helices, oneof which was aligned in sequence (Chan at al., Cell 89, 263-273, 1997;Weissenhorn et al., Nature 387, 426-430, 1997). The prefusion structureof gp41 has, however, resisted atomic-level analysis. Because theprimary structural rearrangement driving membrane fusion is the gp41transition from prefusion to postfusion conformations, the lack of aprefusion gp41 structure has stymied attempts to provide a coherentpicture of the conformational rearrangements the spike undergoes tofacilitate entry.

Here, neutralizing antibodies PGT122 (Walker et al., Nature 477,466-470, 2011) and 35O22 were used to capture the HIV-1 ectodomaintrimer in a mature near-native state. Crystals of the antigen-bindingfragments (Fabs) of these two antibodies were obtained in complex with asoluble, cleaved, Env trimer construct (BG505 SOSIP.664; Sanders et al.,Journal of virology 76, 8875-8889, 2002; Julien et al., PNAS 110,4351-4356, 2013; Sanders, PLoS pathogens 9, e1003618, 2013) and thestructure of this elusive immunological target was determined atatomic-level detail. Analysis of this structure in the context ofpreviously determined gp120 and gp41 structures affords a mechanisticunderstanding of the conformational transitions the ectodomain trimerundergoes to facilitate virus entry. Delineate aggregate parameters ofglycan shielding and genetic variation were determined and a cohortserum was used to determine where the immune system succeeds inrecognizing the HIV-1 ectodomain trimer. Analysis of the matureHIV-1-Env structure and its conformational rearrangements, combined withan understanding of its evasion from and vulnerabilities to the immunesystem, provide an information matrix which can be exploited tomanipulate this critical vaccine target.

Structure determination and overall structure. Atomic-level informationfor virtually all of the HIV-1 Env ectodomain has been obtained asantibody-bound Env complexes (FIG. 17 ). FIG. 17 illustrates structuredetermination as deposited as PDB Accession Nos. 1GGI (Stanfield et al.,PNAS, 90, 6325-6329, 1993), 1GC1 (Kwong et al., Nature, 393, 648-659,1998), 1F58 (Stanfield et al., Structure, 7, 131-142, 1999), 1B03(Tugarinov et al., Nat. Struct. Biol., 6, 331-335, 1999), 2F5B (Pai etal., Patent No. CA2371929), 1TJI (Ofek et al., J. Virol., 78,10724-10737, 2004), 1TZG (Cardoso et al., Immunity, 22, 163-173, 2005),2B4C (Huang et al., Science, 310, 1025-1028, 2005), 2CMR (Luftig et al.,Nat. Struct. Mol. Biol., 13, 740-747, 2006), 2FX7 (Cardoso et al., J.Mol. Biol., 365, 1533-1544, 2007), 3JWD (Pancera et al, PNAS, 107,1166-1171, 2010), 3U2S (McLellan et al., Nature, 480, 336-343, 2011),4G6F (Huang et al., Nature, 491, 406-412, 2012), 4CC8 (Bartesaghi etal., Nat. Struct. Mol. Biol., 20, 1352-1357, 2013), 4NCO (Julien et al.Science 342, 1477-1483, 2013), and 3J5M (Lymunkis et al., Science, 342,1484-1490, 2013).

The recently determined electron microscopy (EM) reconstruction (Lyumkiset al., Science 342, 1484-1490, 2013) and crystal structure (Julien etal., Science 342, 1477-1483, 2013) of a soluble cleaved HIV-1 Env basedon the BG505 SOSIP.664 construct were no exceptions, in particular—whilean artificial disulfide and other modification of the SOSIP.664construct were critical for production of homogeneous, soluble, cleavedtrimers (Ringe et al., PNAS 110, 18256-18261, 2013)—antibody PGT122appeared to facilitate crystallization of a near-native mature state(Julien et al., Science 342, 1477-1483, 2013). Diffraction from crystalsof the PGT122 complex, however, extended to only 4.7-Å resolutionhampering the trace of non-helical regions of gp41 as well as theplacement and registry of side chains (Julien et al., Science 342,1477-1483, 2013). Addition of antibody 35O22 to PGT122-bound viral spikein the membrane-bound virion context showed single-molecule fluorescentresonance energy transfer (smFRET) responses that closely resembled thatof the mature native unliganded ectodomain trimer (FIG. 1 a , FIG. 7 ;Munro et al. Biophysical Journal 104, 415A, 2013). In the context ofcrystallization, addition of 35O22 to the PGT122-BG505 SOSIP.664 complexled to ternary complex crystals in space group P6₃. While diffractionwas anisotropic, we succeeded in collecting ˜3.5-Å data from a singlecrystal (57.2% complete with 2.2 I/σ in the 3.49-3.68 Å shell with 3 I/σmeasurements extending to 2.9 Å along the 6-fold axis) (FIG. 14 ; FIG.18 ). Structure solution by molecular replacement with free structuresof Fab PGT122 (Julien et al., PLoS pathogens 9, e1003342, 201) and Fab35O22 and antibody-bound gp120 (Georgiev et al., Science 340, 751-756,2013) followed by model building and refinement revealed a doubleantibody-bound protomer to occupy the asymmetric unit and led to anRwork/Rfree of 21.35%/24.80%. The final model, comprising PGT122 and35O22 Fabs, residues 31-505 of gp120 (except 185a-186 and 399-410 invariable regions V2 and V4, respectively) and residues 518-664 of gp41(except 548-568) along with 22-N-linked glycans and 10-sulfate ions isshown in FIG. 1B (for clarity, from this point forwards in this Example,residue numbers are cited with a subscript defining the molecule). 35O22interactions with the HIV-1-Env trimer are shown in FIG. 8 , andcomparison of bound vs. unbound Fab structures is shown in FIG. 9 .

Overall, the HIV-1 ectodomain trimer forms a 3-blade propeller, cappedat the membrane-distal apex by trimer association domains withantibodies PGT122 and 35O22 binding to membrane-distal andmembrane-proximal ends, respectively, of the ectodomain trimer (FIG. 10). Protomer interactions occur through the trimer association domains atthe membrane-distal portion of the ectodomain timer and also throughgp41, primarily between helical interactions around the trimer axis. Notrimeric interactions are contributed by the gp120 core: indeed, a cleftor opening is found under the trimer association domains along the3-fold axis where such associations might occur. The trimeric gp41 formsa platform, through which the gp120 termini extend towards the viralmembrane (FIG. 1 b ). Unusually slow signal peptide cleavage, whichkeeps the N terminus of gp120 proximal to the membrane during thefolding, may facilitate gp41 folding.

Prefusion structure of gp41. Prefusion gp41 wraps its hydrophobic corearound extended N- and C-termini-strands of gp120 (FIG. 2 a ). It formsa 4-helix collar comprising helices α6 (Met530_(gp41)-Asn543_(gp1)), α7(Gly572_(gp41)-Ile595_(gp41)—which aligns in sequence with theC-terminal portion of the postfusion HR1 helix), α8(Leu619_(gp41)-Trp623_(gp41)), and α9 (Trp628_(gp41)-Asp664_(gp41)—whichaligns in sequence with the postfusion HR2 helix) (the numbering ofprefusion gp41-helices and -strands continues the nomenclatureestablished for the gp120 subunit, which ends with helix α5 and strandβ26). The first residue of gp41 visible in the electron densitycorresponds to Val518_(gp41), in the fusion peptide. An extended stretchconnects to Leu523_(gp41), which interacts hydrophobically withTrp45_(gp120) and Ile84_(gp120), both of which are part of the7-stranded β-sandwich around which the gp120-inner domain is organized(Pancera PNAS 107, 1166-1171, 2010; Finzi et al., Molecular cell 37,656-667, 2010). The main chain of gp41 follows gp120-strand β0 away fromthe trimer axis towards the viral membrane, until residue Met530_(gp41),where the fold reverses itself and extends through the α6 helix towardsthe trimer axis and away from the viral membrane. Density betweenresidues 547_(gp41) and 569_(gp41) is sparse (FIG. 19 ), and ultimatelyconnects to α7, which forms a coiled-coil with itself around the trimeraxis, extending from the middle of the HIV-1 ectodomain trimer towardsthe viral membrane. At the end of α7 is the gp41-cysteine loop (spannedby the Cys598_(gp41)-Cys604_(gp41) disulfide), whose C-terminal residuesinitiate strand β27 (Leu602_(gp41)-Thr606_(gp41)), which hydrogen bondsin anti-parallel fashion with strand β-4 from the N terminus of gp120.The intersubunit disulfide (‘SOS’) between residues 501_(gp120) and605_(gp41) welds the C terminus of gp120 to the membrane-proximal end ofstrand β-4 (FIG. 2 a ). These membrane-proximal interactions are furtherstabilized by hydrophobic interactions, which gp41 makes with the N andC termini of gp120—such as between Trp35_(gp120) and Pro609_(gp41) andbetween Trp610_(gp41) and Pro498_(gp120). Upon passing the gp120termini, gp41 reaches α8, whose C terminus aligns spatially with the Nterminus of α6. After α8, the α9 helix reverses direction, again wrapspast the N- and C-termini of gp120, before extending horizontally alongthe rim of the ectodomain trimer to reach the gp120 termini from aneighboring protomer.

Topologically, the gp41 subunit completes a single circle around thegp120 termini with the insertion of a hydrophobic prong comprising theside chain of Met530_(gp41) (which is located at the start of α6,proximal to the fusion peptide), into a triple tryptophan-clasp formedby Trp623, (from the end of α8), Trp628_(gp41) (from the start of α9)and Trp631_(gp41) (one turn into α9) (FIG. 2 a insert). The alignment ofdipoles from helices α6 and α8 likely provides electrostaticcomplementarity that help to stabilize the neighboringmethionine-tryptophan clasp.

Within a single protomer, the buried surface between gp41 and gp120totals 5,268 Å², including 216 Å² from glycan-protein interactions (FIG.25 ). A substantial portion of this is hydrophobic: gp41 essentiallywraps its hydrophobic core around the N- and C-termini of gp120 (FIG. 2b ). Trimer interfaces also bury a large surface area (3,138 Å2contributed by each protomer, comprising 1,917 Å² from the gp41-gp41interface, 861 Å² from the gp120-gp120 interface and 360 Å² from thegp120-gp41 interface) (FIGS. 20 and 25 ). Close to the trimer axis,these involve helix α7, as well as the N-terminal portion of thegp41-cysteine loop. Further from the trimer axis, interactions involveα9. Other than interactions of α7, most of the interprotomerinteractions are hydrophilic (FIG. 2 c , FIG. 20B). Overall, theprefusion structure of gp41 as well as its trimeric arrangement appearsto have no close structural relatives in the PDB (FIG. 26 ).

Prefusion to postfusion gp41 transition. To understand theconformational transition from prefusion to postfusion gp41, thegp41-prefusion structure in the near-native HIV-1 Env trimer wascompared with previously determined postfusion structures (FIG. 3 ).Several postfusion gp41 structures have been determined ranging from aminimal, protease-treated, crystal structure (residues556_(gp41)-581_(gp41); 628_(gp41)-661_(gp41); PDB 1AIK; Chan at al.,Cell 89, 263-273, 1997) with 80% sequence identity to BG505 (Wu et al.Journal of virology 80, 835-844, 2006) to a more complete gp41 structure(residues 531_(gp41)-581_(gp41); 624-681_(gp41); PDB 2X7R; Buzon et al.PLoS pathogens 6, e1000880, 2010) and an NMR structure that includes thecysteine loop (residues 539_(gp41)-665_(gp41); PDB 2EZO; Caffrey, M. etal. EMBO 17, 4572-4584 (1998) of the simian immunodeficiency virus(SIV), which shares 48% sequence identity with BG505 (Wu et al. Journalof virology 80, 835-844, 2006) and is substantially similar to the HIV-1structures (less than 1-Å Cα root-mean-square deviation (rmsd) betweenoverlapping residues of 1AIK and 2EZO). To provide comparison with a“complete” postfusion structure, a chimera of HIV-1/SIV structures wasprepared (FIG. 21 ). Distance difference analysis (FIG. 3B) of prefusionand postfusion structures indicated two regions of substantialsimilarity, corresponding to (i) the prefusion α7 helix aligned with theC-terminal half of the postfusion HR1 helix and (ii) the prefusion α9helix aligned with much of the postfusion HR2 helix.

Superposition of prefusion α7 and postfusion HR1 placed residues569_(gp41)-593_(gp41) within 5 Å, with a rmsd of 1.35 Å. For thissuperposition to occur, Ca-movements of over 80 Å are required for thegp41-fusion peptide and α6 helix as well as for the C-terminal portionof the α9 helix. Notably, this superposition preserves the coiled coiltrimeric interaction of both prefusion and postfusion molecules and thuslikely mimics the natural conformational transition that occurs duringmembrane fusion. Meanwhile, superposition of prefusion α9 and postfusionHR2 placed residues 634_(gp41)-664_(gp41) within 5 Å, with a rmsd of3.58 Å; the substantial alignment of the α9 and HR2 helices indicatethat the HR2 helix is mostly preformed in the prefusion structure.

Entry rearrangements of HIV-1 Env. Biosynthesis of HIV-1 Env starts withan uncleaved gp160 trimer. Binding by antibodies PG9 and PGT145 to bothuncleaved and mature Env indicate the trimer association domains at thespike apex likely assume conformations similar to that observed for themature ectodomain trimer (Walker et al., Nature 477, 466-470, 2011;Walker et al. Science 326, 285-289, 2009) (FIG. 27 ). The structure ofgp41 in the uncleaved state remains unknown, but antigenic differenceswith the mature cleaved state (Blattner et al. Immunity 40, 669-680,2014; Falkowska et al. Immunity 40, 657-668, 2014) suggest a distinctgp41 conformation; in the prefusion HIV-1-Env structure, the observed Cterminus of gp120 at residue 505_(gp120) and N terminus of gp41 atresidue 518_(gp1) are 37 Å apart, a distance which cleavage may help theprefusion structure to accommodate. After cleavage, the ectodomaintrimer condenses into the closed near-native mature structure describedhere. In the gp120-inner domain, helix α-1 is formed, and a parallelstrand exists between β3 and β21; in gp41, helix α7 was observed tobegin around residue 571_(gp41). A partially open ectodomain trimerconformation has been reported at 6 Å by EM reconstruction (Bartesaghi,Nature structural & molecular biology 20, 1352-1357, 2013). The trimerassociation domains appear to be displaced from the trimeric axis, andhelical density suggests helix α7 to start several turns earlier,extending ˜20 Å towards the target cell membrane; we modeled theserearrangements with a rigid body motion of 6 degrees of the gp120protomer and by the conversion of ˜15 residues of helix α6 andconnecting stretch into helix α7 (FIG. 3D, middle panel; 15).

The CD4-bound state has been visualized by a number of EMreconstructions (Liu, Nature 455, 109-113, 2008; White et al. PLoSpathogens 6, e1001249, 2010) and atomic-level structures (Kwong et al.Nature 393, 648-659, 1998; Pancera PNAS 107, 1166-1171, 2010). In thisstate, V1V2 separates from V3: V3 points towards the target cell (Huanget al. Science 310, 1025-1028, 2005), and the bridging sheet (Kwong etal. Nature 393, 648-659, 1998) assembles with β2 forming antiparallelhydrogen bonds with β21 (as opposed to the parallel β3-β21 interactionof the near-native mature state; notably, the only parallel β-strand inthe RSV F glycoprotein prefusion structure also changes conformation inRSV F pre- to postfusion transition; McLellan et al. Science 340,1113-1117, 2013). With layer 1 of the inner domain (Finzi et al.,Molecular cell 37, 656-667, 2010), helix α0 forms and Gln428_(gp120) andstrand β21 invert; and in layer 2, inner domain rearrangements includethe swapping of distinct perpendicular interactions of Trp112_(gp120)and Trp427_(gp120) (FIG. 11 ). CD4 binding allows HR2 peptide analogues(such as T20 or C34) to bind (Yuan et al., Journal of virology 78,5448-5457, 2004; and helix α7 can be modeled starting as early as554_(gp41) with Met530_(gp41) still in its membrane-proximal tryptophanclasp (FIG. 22 ), as expected because 35O22 binds the CD4-bound SOSIP(FIGS. 16 and 23 ). It is expected that Env-CCR5 interactions (Huang etal., Science 317, 1930-1934, 2007) bring the CD4-bound state close tothe target cell membrane, where the “de-assembling α6/assembling α7helices” coupled to release of the Met530_(gp41) prong from itstryptophan clasp ultimately amasses the gp41-fusion peptide(s) (FIG. 3d, 2nd panel from right).

At this receptor-bound stage, it is easy to imagine the fusion peptidepenetrating the target cell membrane, while β27 gp41-cysteine loopremains hydrogen bonded to the gp120 termini (and with the C terminus ofthe gp41 ectodomain is in the viral membrane). Rearrangement of gp41 toits postfusion conformation may be triggered by gp120 shedding (Moore etal., Science 250, 1139-1142, 1990), with expulsion of its terminitugging on the gp41-cysteine loop and destabilizing the prefusion gp41core. We note that the three tryptophans that make up thegp41-tryptophan clasp are essential to the folding of the post-fusioncoiled coil, so they appear to be critical in both conformations (FIG.24 ). The MPER region likely associates with a number of lipidsassisting the fusion of viral and target cell membrane.

HIV-1 rearrangements and other type 1 fusion machines. To determinewhether the distinct elements observed in prefusion gp41 were preservedelsewhere, prefusion and postfusion states of other type I fusionmachines from influenza virus (a member of the Orthomyxoviridae familyof viruses; Wilson et al., Nature 289, 366-373, 1981, Bullough et al.,Nature 371, 37-43, 1994), respiratory syncytial virus (RSV;Paramyxoviridae, McLellan et al. Science 340, 1113-1117, 2013, McLellanet al., Journal of virology 85, 7788-7796, 2011), and Ebola virus(Filoviridae, Weissenhorn et al., Molecular cell 2, 605-616, 1998, Leeet al., Nature 454, 177-182, 2008) were examined (FIG. 4 a ). In allcases, a helix was observed in the gp41-prefusion equivalents, whichcorresponds in sequence to the C-terminal portion of the helix, which inthe postfusion conformation, comprises the internal coiled coilcharacteristic of type I fusion machines (FIG. 4 b ). With prefusionmachines from HIV-1, influenza, and Ebola, the nascent prefusion helixadopts a coiled coil; with RSV, a coiled coil assembles immediately Nterminal to the nascent postfusion helix. Despite dramatic differencesin gp120-equivalents, similarity is also observed in the overalltopology of subunit interactions. Notably, all of the gp41-equivalentswrap hydrophobic residues around extended termini (or terminus) of theirgp120-equivalents (FIG. 4 c ). With influenza, it is only the N terminusof the gp120-equivalent (HA1) that is wrapped by the gp41-equivalent(HA2), with the N terminus of HA2 completing about 20% more than asingle encirclement. With RSV, it is also only the N terminus of thegp120 equivalent (F2) that is wrapped by the gp41-equivalent (F1), andthe termini do not have to be expelled to transition to the postfusionform. With Ebola, the gp41-equivalent (gp2) wraps around both N and Ctermini-strands of the gp120-equivalent (gp1), completing about 70% of asingle encirclement. Overall, the similarity in prefusion foldingtopology and in prefusion and post-fusion inner helices observed here,along with the previously observed similarity in postfusion coiled coils(reviewed in Colman et al., Nature reviews. Molecular cell biology 4,309-319, 2003), provide a more general and integrated view of theconformational rearrangements that type 1 fusion machines undergo tofacilitate virus-cell membrane fusion.

Glycan shield and genetic variation of mature unliganded Env. The matureunliganded conformation of HIV-1 Env is the target of most neutralizingantibodies. Substantial detail has already been reported regardingantibody recognition of gp120 in this conformation (Julien et al.,Science 342, 1477-1483, 2013; Lyumkis et al., Science 342, 1484-1490,2013). The newly revealed structure of a near-complete gp120-gp41 Envtrimer provides an opportunity to understand aggregate properties ofglycosylation and variation. Glycan shielding and genetic variation havelong been recognized as mechanisms to avoid recognition by antibody(Wyatt et al., Nature 393, 705-711., 1998). The BG505 SOSIP.664 sequencecontains 28 sequons specifying N-linked glycosylation (including a T332Nmutation). We modeled high mannose glycans (either Man9 or Man5) on eachsequon and calculated accessible surface for radii ranging from 1.4 Å(the radius of a water molecule) to 10 Å (the approximate radius of asingle immunoglobulin domain) (FIG. 12 ). In the Man9-glycosylatedstructure, 29% of the protein surface was solvent accessible, whereasonly 3% of the surface was immunoglobulin-domain accessible. Bycontrast, with the fusion glycoproteins from influenza and RSV, 14% and48%, respectively, of these surfaces were immunoglobulin-domainaccessible (FIG. 5 a ).

In terms of genetic variation, the per-residue Shannon entropy of 3,943sequences of HIV-1 was calculated (FIG. 5 b ). Approximately 50% of thesurface was shown to have a variability of greater than 10%, a degree ofsurface variation shared by influenza, but not by RSV. When glycanshielding and genetic variation were combined, only ˜2% of the surfacewas immunoglobulin accessible with a variability of less than 10% (FIG.5 c , upper panels); much of this conserved surface occurred at themembrane-proximal “base” of the ectodomain trimer, which is expected tobe sterically occluded by the viral membrane. To determine how thisfully assembled shield compared to other conformations, theimmunoglobulin accessibility of the CD4-bound conformation was alsoassessed (FIG. 5 c ). Notably the CD4-bound conformation showedsubstantially higher percentage of glycan-free, conserved surface,providing insight into the greater ease by which antibodies reactivewith the CD4-bound conformation are elicited—and by contrast, thedifficulty in eliciting broadly neutralizing antibodies against thevariable, glycan-covered mature state.

Serologic recognition of mature Env. Despite the multiple mechanisms ofevasion shielding mature HIV-1 Env, potent broadly neutralizingantibodies do develop (Hraber et al. AIDS 28, 163-169, 2014). Many ofthese, including the PGT122 and 35O22 co-crystallized here, requireN-linked glycosylation to bind; indeed, 35O22 utilizes a new mode ofglycan recognition, involving a framework 3 insertion to create a “bowl”that cups glycan N88_(gp120) (FIG. 8 ). The near-native mature prefusionstructure of HIV-1 Env allows us to map known epitopes (FIG. 6 a ) andto compare the recognition of broadly neutralizing HIV-1 antibodies,with those capable of neutralizing influenza virus and RSV. Notably, theepitopes for broadly neutralizing HIV-1 antibodies were significantlymore glycosylated and variable (FIG. 6 b ).

To determine the location and prevalence of effective humoral responses,a serological analysis was used that determined sites of HIV-1vulnerability to antibody based on serum neutralization of a panel ofdiverse HIV-1 isolates (Georgiev et al., Science 340, 751-756, 2013).Sera from a cohort that had been infected for 2-3 years as well as serafrom a cohort of donors that had been infected for more than 5 yearswere assessed on a panel of 21 diverse HIV-1 isolates, and theneutralization phenotypes assigned to 12 prototypicantibody-neutralization fingerprints (FIG. 6 c , FIG. 13 ). We thenmapped the responses to the surface of the near-native mature HIV-1-Envectodomain trimer (FIG. 6 d ). The most prevalent response correspondedto the glycan V3 epitope epitomized by antibody PGT128. CD4-bindingsite-directed responses and also V1V2-directed responses were prevalent.Overall, responses to both cohorts were highly correlated indicatinglittle evolution in the location or prevalence of effective neutralizingresponses between 2-3 years and 5+ years. Notably, when mapping Envsites of vulnerability to neutralizing antibody, the majority ofprevalent sites corresponded to Env surfaces covered by N-linkedglycosylation and/or of high sequence variability. Overall, mapping ofthe location of cohort humoral responses directly visualized prevalenttargets of vaccine relevance (FIG. 6 e ).

Viral evasion and immune recognition. In addition to merging virus andhost cell membranes as an essential step in entry, viral fusion machinesmust contend with antibody-mediated neutralization. With RSV, peakinfection occurs at 6-12 months of life, when maternal antibodies wane;with influenza virus, natural infection elicits strain-specificantibodies, and evasion occurs seasonally on a global scale. HIV-1,however, confronts the immune system in each individual directly, oftenpresenting high titers of Env antigens over years of chronic infection.These differences in evasion are reflected in structural difference inthe fusion machines. The structure of the HIV-1-Env ectodomain trimerrevealed here allows the molecular trickery behind single spike entry(Yang et al., Journal of virology 79, 12132-12147, 2005), glycanshielding (Wei, X. et al. Antibody neutralization and escape by HIV-1.Nature 422, 307-312, 2003), and conformational masking (Kwong et al.Nature 420, 678-682, 2002) to be visualized at the atomic level (FIG. 31). Thus, avoidance of antibody avidity through the ability of a singleHIV-1 spike to fuse viral and target cell membranes (Yang et al.,Journal of virology 79, 12132-12147, 2005) is likely assisted by themembrane-proximity of the co-receptor and the membrane-associating MPERregions (FIG. 3 ); despite these differences, the HIV-1-Env ectodomaintrimer appears to share mechanism and topology with other type 1 fusionmachines (FIG. 4 ). In terms of glycan shielding (Wei, X. et al.Antibody neutralization and escape by HIV-1. Nature 422, 307-312, 2003),we have modeled the structure of a fully assembled glycan shield for atier II transmitted founder virus (Wu et al. Journal of virology 80,835-844, 2006) (FIG. 5 ). While glycan masking appears complete at theHIV-1-spike apex, closer to the membrane substantial “holes” areobserved. And with conformational masking (Kwong et al. Nature 420,678-682, 2002), evasion is optimal for the prefusion mature closedstate, with CD4-binding unmasking conserved glycan-free surfaces (FIG. 5c ). Despite extraordinary glycosylation and sequence variation, thehuman immune system appears up to the challenge of generating broadlyneutralizing antibodies (FIG. 6 ). It is noted that recognition ofglycosylation appears to be a trait common only to broadly neutralizingHIV-1 antibodies, although broad influenza virus-neutralizing antibodiesdo appear to tolerate epitope-sequence variation (FIG. 6 b ). Thestructure of the HIV-1-Env ectodomain trimer described here thus revealsnot only commonalities in entry and evasion with other type 1 fusionmachines, but also commonalities in recognition by the human immunesystem.

Methods

BG505 SOSIP.664 expression and purification. The crystallized HIV-1-Envconstruct from strain BG505 was synthesized as described in (Julien etal., Science, 342, 1477-1483, 2013; Julien et al., Proc. Nat'l. Acad.Sci. U.S.A., 110, 4351-4356, 2013; Sanders et al., PLoS pathogens, 9,e1003618, 2013), using BG505 GenBank® Acc. Nos., using BG505 GenBankaccession numbers ABA61516 and DQ208458 (Wu et al. Journal of virology80, 835-844, 2006), including the “SOS” mutations (A501C, T605C), theisoleucine to proline mutation at residue 559 (1559P), and the glycansite at residue 332 (T332N); mutating the cleavage site to 6R (REKR toRRRRR); and truncating the C terminus to residue 664 (all HIV-1 Envnumbering according to the HX nomenclature). This construct is referredto as BG505 SOSIP.664 herein.

The construct was cotransfected with furin in HEK 293 S GnTI−/− cellsusing 600 μgs plasmid DNA and 150 μgs of furin as described previously(Sanders, PLoS pathogens 9, e1003618, 2013). Transfection supernatantswere harvested after 7 days, and passed over either a 2G12 antibody- orVRC01 antibody-affinity column. After washing with PBS, bound proteinswere eluted with 3M MgCl₂, 10 mM Tris pH 8.0. The eluate wasconcentrated to less than 5 ml with Centricon-70 and applied to aSuperdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl,0.02% azide. The peak corresponding to trimeric HIV-1 Env wasidentified, pooled, and concentrated or flash-frozen in liquid nitrogenand stored at −80° C.

Fab expression and purification. PGT122 and 35O22 IgGs were expressed aspreviously described (McLellan et al., Nature 480, 336-343, 2011). Heavychain plasmids containing an HRV3C cleavage site after Lys 218 in thehinge region were co-transfected with light chain plasmids in 293F(35O22) or GnTI−/− (PGT122, which is glycosylated) using TrueFect-Maxtransfection reagent (United Biosystems) according to manufacturer'sprotocol. Cultures were fed with fresh 293FreeStyle media (LifeTechnologies) 4 h post-transfection and with HyClone SFM4HEK293 enrichedmedium (HyClone) containing valproic acid (4 mM final concentration) 24h after transfection. Cultures were then incubated at 33° C. for 6 days,and supernatants harvested and passed over a protein A affinity column.After PBS wash and low pH elution, pH of eluate was neutralized with 1MTris pH 8.5. Fabs were obtained using HRV3C digestion and collectingflow-thru from protein A column to remove Fc fraction. Fabs were furtherpurified over Superdex 200 in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02%azide.

Ternary complex preparation. PGT122 and 35O22 Fabs were added to asolution of purified trimeric BG505 SOSIP.664 in 5 fold molar excess for30 min at room temperature (RT). The complex was then partiallydeglycosylated by adding Endo H (50 μl) for 1 hour at RT in the gelfiltration buffer. The complex was then purified over gel filtrationequilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% azide. Fractionswere pooled, concentrated down to 5-10 OD₂₈₀/mL and used immediately forcrystal screening or flash frozen in liquid nitrogen and kept at −80° C.until further use.

Crystallization screening. The ternary complex was screened forcrystallization using 572 conditions from Hampton, Wizard andPrecipitant Synergy (Majeed, S. et al. Structure 11, 1061-1070 (2003)screens using a Cartesian Honeybee crystallization robot as describedpreviously (McLellan et al., Nature 480, 336-343, 2011) and a mosquitorobot using 0.1 μl of reservoir solution and 0.1 μl of protein solution.Crystals suitable for structural determination grew in 0.2M Li₂SO₄,6.65% PEG 1500, 20% isopropanol and 0.1M sodium acetate pH 5.5. Crystalswere reproduced in hanging droplets containing 0.5 μl of reservoirsolution and 0.5 μl of protein solution. The final crystals wereobtained in 16% isopropanol, 5.32% PEG 1500, 0.2M Li₂SO₄, 0.1M Naacetate pH 5.5. The crystals were cryoprotected in a solution of 15%2R3R-butanediol, 5% isopropanol in paratone N and data were collected ata wavelength of 1.00 Å at the SER-CAT beamline ID-22 (Advanced PhotonSource, Argonne National Laboratory).

X-ray data collection, structure solution and model building.Diffraction data were processed with the HKL2000 suite (Otwinowski andMinor, Meth. Enzymol., 276, 307-326, 1997). The data were corrected foranisotropy by services.mbi.ucla.edu/anisoscale/ with truncations to 3.5Å, 3.5 Å, 3.1 Å along a, b, and c axes, respectively. Structure solutionwas obtained with Phaser using gp120 (PDB ID: 4J6R; Georgiev et al.,Science 340, 751-756, 2013), PGT122 (PDB ID: 4JY5; Julien et al., PLoSpathogens 9, e1003342, 2013) and 35O22Fv as search models. Refinementwas carried out with Phenix (Adams et al. J Synchrotron Radiat, 11,53-55, 2004) imposing PGT122, 35O22 and gp120 model-based refinementrestraint during initial round of refinement. Model building was carriedout with Coot (Emsley and Cowtan, Acta crystallographica. Section D,Biological crystallography, 60, 2126-2132, 2004). The Ramachandran plotas determined by MOLPROBITY (Davis et al., Nucleic Acids Res, 32,W615-619, 2004) showed 92.66% of all residues in favored regions and99.03% of all residues in allowed regions. Data collection andrefinement statistics are shown in FIG. 14 .

smFRET. Peptides for site-specific fluorescent labeling were insertedinto HIV-1_(JR-FL) gp160 at positions that did not interfere with Envfunction by overlap extension PCR. Tagged virus was purified andlabelled with Cy3B and Cy5(4S)COT fluorophores, and surface immobilizedfor imaging via total internal reflection fluorescence (TIRF) microscopyas described (Munro et al. Biophysical Journal 104, 415A, 2013).Labelled virus was pre-incubated for 30 min with 0.1 mg/ml PGT122 or35O22, or with both PGT122 and 35O22 prior to imaging. Fluorescencetrajectories were acquired at 25 frames/s. Traces that presentedanticorrelated fluctuations in fluorescence intensity, indicative ofFRET, were identified and compiled into histograms. Histograms were fitto the sum of three Gaussian distributions in Matlab. smFRET revealedthat HIV-1 Env is conformationally dynamic, transitioning between threedistinct conformations. Response to various ligands identified thelow-FRET conformation as the predominant population of mature prefusionunliganded HIV-1 Env; intermediate- and high-FRET conformationspredominant in the presence of CD4 and CD4-induced antibodies (Munro etal. Biophysical Journal 104, 415A, 2013).

Binding studies using biolayer interferometry. A fortéBio Octet Red384instrument was used to measure binding of BG505 SOSIP. 664 and BG505gp120 molecules to neutralizing antibodies (VRC01, VRC03, b6, b12, F105,PGT122, PGT128, PGT135, 2G12, 8ANC195, 17b, 2.2C, 412d, PG9, PGT145,VRC26.09, 35O22, PGT151) and CD4 Ig. All the assays were performed withagitation set to 1,000 rpm in phosphate-buffered saline (PBS) buffersupplemented with 1% bovine serum albumin (BSA) in order to minimizenonspecific interactions. The final volume for all the solutions was40-50 μl/well. Assays were performed at 30° C. in solid blacktilted-bottom 384-well plates (Geiger Bio-One). Human antibodies (40-50μg/ml) in PBS buffer was used to load anti-human IgG Fc capture (AHC)probes for 300 s. Typical capture levels were between 1 and 1.5 nm, andvariability within a row of eight tips did not exceed 0.1 nm. Biosensortips were then equilibrated for 180 s in PBS/l % BSA buffer prior tobinding assessment of the BG505 SOSIP.664 and BG505 gp120 molecules insolution for 300 s; binding was then allowed to dissociate for 300 s.Parallel correction to subtract systematic baseline drift was carriedout by subtracting the measurements recorded for a sensor withoutmonoclonal antibody incubated in PBS/1% BSA. Data analysis were carriedout using Octet software, version 8.0.

Difference distance analysis. Difference distance matrices were producedby distance sorting atom positions and plotting with the program DDMP(Nishikawa et al., J. Physical Society of Japan 32, 1331-1337 (1972).

Surface plasmon resonance analysis. Affinities and kinetics of bindingof antibodies 35O22 and PGT151 to BG505 SOSIP.664 soluble trimer wereassessed by surface plasmon resonance on a Biacore T-200 (GE Healthcare)at 20° C. with buffer HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mMEDTA, and 0.05% surfactant P-20). In general, mouse anti-human Fcantibody was first immobilized onto two flow cells on a CM5 chip at˜10000 response units (RU) with standard amine coupling protocol (GEHealthcare). Either CD4-Ig, 2G12 IgG or 17b IgG was then captured onboth flow cells by flowing over a 200 nM solution at 5 μl/min flow ratefor two minutes. This was followed by a 1-minute injection of 1 μM humanFc on both flow cells to block unliganded mouse anti-human Fc antibody.The captured 2G12, CD4 or 17b were used to immobilize BG505 SOSIP.664trimer on only one flow cell, with no trimer captured on the other flowcell (reference cell). For capturing with 2G12 or CD4-Ig, 500 nM ofunliganded trimer was used, whereas, a complex of 500 nM trimer+1500 nMsCD4 was used for capturing with 17b. Antibody Fab fragments at 2-folddilutions starting from 885 nM, 600 nM and 460 nM for 35O22, PGT151 andPGT145, respectively, were injected over the captured trimer channel andthe reference channel at a flow rate of 50 μl/min for 2 minutes andallowed to dissociate for 3-30 minutes depending on the rate ofdissociation of each interaction. The cells were regenerated with two 10μl injections of 3.0 M MgCl₂ at a flow rate of 100 μl/min. Blanksensorgrams were obtained by injection of same volume of HBS-EP+bufferin place of antibody Fab fragments. Sensorgrams of the concentrationseries were corrected with corresponding blank curves and fittedglobally with Biacore T200 evaluation software using a 1:1 Langmuirmodel of binding. The stoichiometry of binding of antibodies to thetrimer were estimated by normalizing the Rmax values to the amount oftrimer captured and performing linear regression analysis using the Rmaxvalues for the antibodies with known stoichiometries.

Modeling of missing loops, side chains, and the N-linked glycan shield.Missing loops not defined in the HIV-1-Env trimer crystal structure weremodeled using Loopy (Xiang et al., Proc. Nat'l. Acad. Sci. U.S.A., 99,7432-7437, 2002). The Missing side chains were modeled with Scap (Xiangand Honig, J Mol. Biol., 311, 421-430, 2001).

To model the N-linked glycan shield, we first determined all possibleN-linked sequons in the HIV-1 Env trimer structure. A single asparagineresidue in each sequon was targeted for computational N-linked glycanaddition using a series of oligmannose 9 rotamer libraries at differentresolutions. In constructing the rotamer libraries, the asparagine sidechain rotamers were also considered. To avoid a combinatorial explosionin the search space, select torsion angles in the oligomannose 9 rotamerlibraries were allowed to vary in increments between 30-60 degrees. Anoverlap factor (ofac) was used to screen for clashes between the sugarmoieties and the trimer structure. The ofac between two nonbonded atomsis defined as the distance between two atoms divided by the sum of theirvan der Waal's radii. For the modeling carried out here, the ofac wasset to a value of 0.60. For sterically occluded positions, the ofac wasset to 0.55. To remove steric bumps between sugar moieties, all modelswere subjected to 100 cycles of conjugate gradient energy minimizationusing the GLYCAM (Kirschner et al. Journal of computational chemistry29, 622-655 (2008)) force field in Amber12 (Cornell J. Am. Chem. Soc.117, 5179-5197, 1995), with a distance-dependent dielectric.

Mapping sequence variability onto trimer structure. For each of HIV-1Env, influenza HA, and RSV F, residue sequence variability was computedas the Shannon entropy for each residue position, based onrepresentative sets of 3943 HIV-1 strains, 4467 influenza strains, and212 RSV strains, respectively. Residues were colored based on thecomputed entropy values, on a scale of white (conserved) to purple(variable).

Serum neutralization fingerprinting analysis. The prevalence ofeffective neutralizing responses against HIV-1 Env in cohorts from 2-3and 5+ years post-infection was estimated using a neutralizationfingerprinting approach, as described previously (Georgiev et al.,Science, 340, 751-756, 2013). Briefly, serum neutralization over a setof 21 diverse viral strains was compared to neutralization of the sameviruses by a set of broadly neutralizing antibodies grouped into 12epitope-specific antibody clusters. For each serum, the relativeprevalence of each of the 12 antibody specificities was estimated byrepresenting serum neutralization as a linear combination of themonoclonal specificities, with prevalence values of 0.2 deemed aspositive. Sera with less than 30% breadth on the 21-virus panel as wellas sera with high residual values from the computation (data not shown)were not included in the analysis. For mapping prevalence values ontothe BG505 structure, residues part of multiple antibody epitopes werecolored according to the respective antibody specificity with thehighest prevalence in the 5+ years cohort. Antibody neutralization wasmeasured using single-round-of-infection HIV-1 Env-pseudoviruses andTZM-bl target cells, as described previously (Li et al., J. Virol., 79,10108-10125, 2005). Neutralization curves were fit by nonlinearregression using a 5-parameter hill slope equation as previouslydescribed (Li et al., J. Virol., 79, 10108-10125, 2005).

Patient information. In the CHAVI 001 cohort, high-risk subjects werescreened for HIV-1 infection by ELISA, Western blotting, and plasma RNAto recruit individuals with acute HIV infection, who were then followedfor ˜2 years until plasma neutralization breadth developed (Tomaras etal., J. Virol., 82, 12449-12463, 2008). In addition, a group ofindividuals were enrolled in the CHAVI 001 or CHAVI 008 cohorts who werechronically infected with HIV-1 strains clade A, B or C, and werescreened for plasma neutralization breadth. The trial participants wereenrolled at sites in Tanzania, South Africa, Malawi, the United States,and the United Kingdom (Tomaras et la., J. Virol., 85, 11502-11519,2011). Both CHAVI001 and CHAVI008 protocols were approved by theinstitutional review boards of each of the participating institutionswhere blood samples were received or processed for analysis.

Epitope analysis for HIV-1 Env, influenza HA, and RSV F antibodies.Glycan usage and average residue entropy were calculated for eightrepresentative HIV-1 Env (VRC01, b12, CD4, HJ16, 8ANC195, PG9, PGT122,2G12, and 35O22), four representative influenza HA (2D1, C05, F10, andCR8043), and three representative RSV F (D25, Motavizumab, and 101F)epitopes based on their respective crystal structures. The selection ofthe flu antibodies was done as follows: F10 (stem targeting) and C05(head targeting) were selected based on their cross-neutralizing abilityfor group 1 and group 2 of influenza A. CR8043 (group 2 specific) and2D1 (H1 specific), which targets distinct regions from F10 and C05 atthe stem and head of the HA respectively, were also selected for epitopeanalysis. An antigen residue was defined as an epitope residue if it hada non-zero BSA in the crystal structure. The fraction of glycan surfacearea in an epitope was calculated as the buried surface area of epitopeglycans divided by the buried surface area of the full epitope.Mann-Whitney test was used to quantify the statistical differencebetween glycan fraction or average residue entropy for HIV-1 vs.influenza or RSV antibody epitopes.

Figures. Structure figures were prepared using PYMOL (The PyMOLMolecular Graphics System, DeLano Scientific, San Carlos, Calif., 2002).

Interfaces. Interactive surfaces were obtained from PISA(ebi.ac.uk/pdbe/pisa/).

Example 2 Crystal Structure of Unliganded HIV-1 Env Trimer in thePrefusion Mature Closed Conformation and Stabilization of the IV-1 EnvEctodomain in a Prefusion Mature Closed Conformation

This example illustrates the three dimensional structure of the BG505SOSIP.664 trimer in the prefusion mature closed conformation when notbound by neutralizing antibody. Additionally, this example illustratesexemplary HIV-1 Env ectodomains stabilized in a prefusion mature closedconformation. The crystal structure of the HIV-1 Env ectodomain incomplex with the PGT122 and 35O22 Fabs (i.e., in a prefusion matureclosed conformation) or in unliganded (without bound antibody) comparedto the structure of HIV-1 Env in the CD4 bound conformation showsdramatic structural rearrangements in both the membrane-proximal andmembrane-distal regions, providing guidance for the stabilization of themature closed conformation of HIV-1 Env.

The structure of the unliganded HIV-1 Env ectodomain trimer issubstantially identical to HIV-1 Env in the PGT122/35O22-bound BG505SOSIP structure (discussed in Example 1) with rmsd of Cα ˜1.1 Å. Thisfinding confirms that the HIV-1 Env ectodomain is not significantlydistorted by binding to PGT122 and 35O22 antibodies, and therefore, thatthe structure disclosed in Example 1 provides an accurate view of theHIV-1 Env ectodomain in the prefusion mature closed conformation.

As the sole viral antigen on the HIV-1-virion surface, trimeric Env—andits gp120 and gp41 subunits—have been the focus of extensive vaccineefforts (Rerks-Ngarm et al., N Engl J Med, 361, 2209-2220, 2009; Flynnet al., J Infect Dis, 191, 654-665, 2005). These have been stymied,however, by unfavorable Env properties including substantialconformational diversity (Kwong et al., Nature, 420, 678-682, 2002).While a near-native soluble Env trimer (BG505 SOSIP.664) has beendeveloped, which is preferentially recognized by broadly neutralizingantibodies (Binley et al., J Virol., 74, 627-643, 2000; Sanders et al.,PLoS pathogens 9, e1003618, 2013; Sanders et al., J Virol, 76,8875-8889, 2002), this trimer can be triggered by the CD4 receptor toexpose epitopes recognized by ineffective antibodies, and aconformationally fixed trimer remains a key goal for vaccine designers.Here the crystal structure at 3.7 Å resolution of the unligandedSOSIP.664 trimer is presented, its structural compatibility withEnv-reactive antibodies is characterized, and structure-based design isused to fix its conformation. The unliganded SOSIP.664 trimer assumed aclosed structure, highly similar to antibody-bound structures (Example1; Julien et al, Science, 342, 1477-1483, 2013; Lyumkis et al., Science342, 1484-1490, 2013), which epitope analysis revealed to bestructurally compatible with broadly neutralizing antibodies, but notineffective ones. Structural compatibility correlated with bindingantigenicity, except for ineffective antibodies directed to CD4-inducedepitopes. Structure-based design yielded conformationally fixedvariants, including a 201-433 double cysteine (DS) mutant, with improvedspecificity for broadly neutralizing antibodies. The DS-SOSIP.664 mutantretained nanomolar affinity for CD4, with which it formed a newstructural state: a closed trimer bound by a single CD4 without thetypical antigenic hallmarks of CD4 induction. This new structural stateappeared to be an obligatory intermediate between the unliganded closedstate and an activated state recognized by multiple CD4s andco-receptor. Conformational fixation—enabled by antigenicity-guidedstructural design—can thus be used to delineate mechanistic states andto improve Env-antigenic specificity, with DS-Env trimers fixed in theunliganded closed state defining a new generation of vaccine immunogens.

HIV-1 uses multiple mechanisms to evade the immune system, and thesehave stymied the development of an effective vaccine. Onemechanism—conformational masking (Kwong et al., Nature, 420, 678-682,2002)—hides the vulnerable shape of trimeric Env recognized by broadlyneutralizing antibodies via structural rearrangements that exposeimmunodominant Env epitopes recognized by non- or poorly neutralizingantibodies. A potential solution is to determine the structure of thevulnerable conformation of Env and to use this structural informationand protein design to stabilize or fix the vulnerable shape. Definitionof the structure of trimeric HIV-1 Env in its vulnerable shape has beenaccomplished at increasing resolution by crystallography andcryo-electron microscopy (Example 1; Julien et al, Science, 342,1477-1483, 2013; Lyumkis et al, Science 342, 1484-1490, 2013; Liu etal., Nature 455, 109-113, 2008). These studies have culminated inatomic-level structures of antibody-bound forms of a soluble,near-native trimer mimic, named BG505 SOSIP.664 for HIV-1 strain (BG505,Wu et al., J Virol, 80, 835-844, 2006) and stabilizing mutations(SOSIP.664, Binley et al., J Virol., 74, 627-643, 2000; Sanders et al,PLoS pathogens 9, e1003618, 2013; Sanders et al., J Virol., 76,8875-8889, 2002). Antibodies, however, can influence conformation, andstructures of the Env gp120 subunit can differ substantially whenunliganded or antibody-bound (FIG. 32A). It was thus sought to determineand to fix the structure of the unliganded HIV-1 Env trimer. Asparse-matrix approach was used to crystallize an endoglycosidaseH-treated BG505 SOSIP.664 trimer from a PEG 400-PEG 3,350 precipitantmixture (Majeed et al., Structure, 11, 1061-1070, 2003). Diffractiondata extended to 3.7-Å resolution, and structure solution and refinementyielded Rwork/Rfree of 26.1%/28.3%.

Overall, despite differences in glycosylation and lattice packing, thestructure of the unliganded trimer assumed a closed conformation, whichwas remarkably similar to antibody-bound trimers (Example 1; Julien etal., Science, 342, 1477-1483, 2013; Lyumkis et al., Science 342,1484-1490, 2013), with an overall root-mean-square deviation (RMSD) inCα positions of less than 1 Å—substantially lower than observed withmonomeric gp120 (FIG. 32A). To determine whether the unliganded closedstructure was an appropriate vaccine template, structurally specific foreffective HIV-1-neutralizing antibodies and incompatible with non- orpoorly neutralizing antibodies, it was first sought to categorizeantibodies by their functional efficacy (FIG. 32B). Broadly neutralizingantibodies were defined as those of greater than 35% breadth on adiverse panel of 170 isolates, with ineffective antibodies of less than15% breadth. Antibodies b12, 35O22 and PGT135 were close to the cutofffor the broadly neutralizing category; and while some antibodies such asthe V3-directed 447-52D showed clade-specific breadth (447-52Dneutralizes over 20% of clade B isolates), 447-52D was nonethelesscategorized as ineffective because its overall breadth was only 12%. Theunliganded closed structure was analyzed for its structuralcompatibility with antibody epitopes—most determined structurally in thecontext of monomeric gp120 or peptide epitope—based on two measures:antibody-volume overlap and epitope RMSD (FIG. 32C). Antibody-volumeoverlap correlated strongly with neutralization breadth (p=0.0020).Epitope RMSD trended with breadth, but did not achieve statisticalsignificance (FIG. 32D). An antibody structural compatibility score(ASC), which combined both overlap and RMSD, did achieve significance(p=0.0069) (FIG. 32D).

The unliganded closed structure was compatible with the epitopes for allbroadly neutralizing epitopes, except those of the membrane-proximalexternal region, which recognize epitopes downstream of residue 664, andof antibodies b1214 and CH10315, with CH103 exceeding a 2 Å threshold ofepitope similarity and with b12 exceeding a volume threshold of 500 Å3(FIG. 32D, left). In light of the poor RMSD correlation with trimerstructure (FIG. 32D), the RMSD threshold was somewhat arbitrary.Nonetheless, the specific incompatibility of these moderately effectiveCD4-binding site antibodies suggest that movement of residues of theCD4-binding site could occur relative to the unliganded closed trimerand still achieve moderate neutralization breadth; indeed, inducedtrimer movements have been observed for b12 (which binds poorly to theBG505 SOSIP.664 trimer, Sanders et al., PLoS pathogens 9, e1003618,2013) by electron microscopy (Liu et al., Nature 455, 109-113, 2008) andhydrogendeuterium exchange. By contrast, none of the epitopes for non-or poorly-neutralizing antibodies were structurally compatible with theunliganded closed structure (FIG. 32D, right).

These results indicate the unliganded closed trimer to be structurallyspecific for neutralizing antibodies. Structural specificity as measuredby epitope compatibility, however, is only one of the requirements of anappropriate vaccine template: antigenic specificity as measured bybinding to broadly neutralizing and not ineffective antibodies is alsocrucial. The BG505 SOSIP.664 had previously been shown by Moore,Sanders, and colleagues to be antigenically specific for broadlyneutralizing antibodies, though binding to poorly neutralizingantibodies directed at the V3 loop was reported (Sanders et al, PLoSpathogens 9, e1003618, 2013). Negative selection by ineffectiveantibodies such as the V3-directed antibody 447-52D17 to removeaberrantly folded molecules was used (FIG. 36 ). However, even afterV3-antibody negative selection, CD4 triggering efficiently exposed V3epitopes (Mbah et al., J Virol, 75, 7785-7788, 2001) as well as bridgingsheet epitopes (Kwong et al, Nature 393, 648-659, 1998) recognized byantibodies like 17b (Thali et al., J Virol., 67, 3978-3988, 1993) (FIG.33A; FIGS. 37-40 ). Overall, while structural compatibility (andneutralization breadth) generally correlated with antibody binding, inthe presence of CD4, this correlation was lost (FIG. 33A; FIG. 37 ).Notably, CD4 triggered ineffective antibodies so that their averagebinding was higher than for broadly neutralizing ones (FIG. 33A, right).CD4 triggering makes BG505 SOSIP.664 less desirable as an immunogen: inprimates, it would bind CD4 in vivo and would thus be predicted toelicit ineffective antibodies against the highly immunogenic CD4-inducedepitopes.

To fix the unliganded closed state and to prevent CD4 triggering,regions of the unliganded closed structure that moved upon CD4 bindingwere analyzed to identify cavity-filling hydrophobic substitutions,side-chain pairs capable of forming disulfide bonds, and positions wherethe introduction of a proline would be compatible with only theunliganded closed structure of Env, but not its receptor-boundconformation (FIG. 33B, insets). These substitutions were engineeredinto BG505 SOSIP.664, co-expressed with furin in a 96-well transfectionformat (McLellan et al., Science 342, 592-598, 2013) and assessedsupernatants on an antigenic panel, comprising broadly neutralizingantibodies PGT122 (Walker et al., Nature 477, 466-470, 2011) and VRC01(Wu et al., Science 329, 856-861, 2010), quaternary-specific broadlyneutralizing antibodies PGT145 (Walker et al, Nature 477, 466-470, 2011)and CAP256-VRC26 (Doria-Rose et al., Nature, 509, 55-62, 2014), andpoorly neutralizing antibodies F105 and 17b, with the latter testedalone and in the presence of CD4. Several different constructs weretested (FIG. 42 ). Several promising constructs were purified andanalyzed for gp120-gp41 cleavage and oligomeric heterogeneity (FIG. 36 )and by meso-scale discovery-electrochemiluminescence immunoassay(MSD-ECLIA) for recognition on a more comprehensive panel ofHIV-1-reactive antibodies (FIG. 33C and FIG. 37 ). One cavity-fillingalteration, Y191W, retained recognition of broadly neutralizingantibodies, but only moderately reduced the binding of antibody 17b,while two proline substitutions, Q432P and A433P, showed improvedantigenic specificity. A 201C-433C double cysteine (DS) mutant showedvirtually no antibody 17b recognition, even in the presence of CD4,while retaining strong recognition of antibody PGT145 and increasing therecognition of antibody CAP256-VRC26 (FIG. 33C). While A433P showedbetter recognition for broadly neutralizing antibodies compared to201C-433C, the temporal stability of A433P was found to be lower thanthat of both BG505 SOSIP.664 and 201C-433C, with 201C-433C exhibitinghighest temporal stability (FIG. 37 ). Notably, with the 201C-433C DSvariant, structural compatibility (and neutralization breadth)correlated with antibody binding, even in the presence of CD4 (FIG. 33D;FIG. 37 ). Modeling of the DS substitution indicated a 201C-433Cdisulfide to be incompatible with the CD4-bound state, where α-carbons(Cα) of residues 201 and 433 are 9.4 Å apart, separated by a strand ofthe bridging sheet (Kwong et al., Nature 393, 648-659, 1998), and the V3loop is fully exposed (Huang et al. Science 310, 1025-1028, 2005) (FIG.33E). By contrast, the 201C-433C substitutions are expected to form adisulfide in the unliganded closed trimer, and indeed the unligandedBG505 SOSIP.664 201C-433C exhibited a 6.1° C. increase inthermostability (to 73.1° C.) relative to the parent SOSIP.664 (FIG.33F).

These results indicate the 201C-433C ‘DS’ variant of BG505 SOSIP.664(termed “DS-SOSIP.664”) is not triggered by CD4. To define theinteraction of the DS-SOSIP.664 variant with CD4, surface plasmonresonance (SPR) was used (FIG. 34A). The DS-SOSIP.664 recognized CD4with a similar on-rate as the parent SOSIP.664, but with ˜10-fold fasteroff-rate, resulting in a ˜10-fold reduction in K_(D) relative toSOSIP.664 (FIG. 34A). To test for CD4 triggering over a longer timescale, both DS-SOSIP.664 and parent SOSIP.664 were incubated for 100 hin the presence of CD4, and SPR readout of 17b and 3074 epitopes wasused to assess triggering. With the parent SOSIP.664, CD4 induced a slowtransition to a state with bridging sheet formed (tin of 3.3±0.7 h forantibody 17b) and V3 loop exposed (t1/2 of 4.2±1.0 h for antibody307426) (FIG. 34B and FIG. 40 ). With DS-SOSIP.664, triggering by CD4 ofbridging sheet or V3 was not observed over the entire 100 h time course(FIG. 34B). To define the stoichiometry of CD4 interaction,sedimentation equilibrium analytical ultracentrifugation of parent andDS-SOSIP.664 variants in the presence of excess CD4 was used. Molecularweights consistent with the parent SOSIP.664 binding two to three CD4sand the DS-SOSIP.664 variant binding only one CD4 were observed (FIG.34C and FIG. 41 ).

DS-SOSIP.664 can thus capture Env in a single CD4-bound state. To obtainstructural information on this single CD4 bound state, thehydrogen-deuterium exchange (HDX) of DS-SOSIP.664 with and without CD4was characterized. Without CD4, the hydrogen-deuterium exchange ofDS-SOSIP.664 appeared similar to the exchange of the parent SOSIP.664(FIG. 34D); with CD4, the gp120 inner domain, the bridging sheet, andgp41 showed little change upon the addition of soluble CD4 (FIG. 34D).The V2, V3 and the stem of V1 showed a response to CD4, consistent withthe slightly increase exposure of V3 epitope observed by MSD-ECLIA (FIG.33D), but this was substantially less than observed for the parentSOSIP.664. The single CD4-bound DS-SOSIP.664 thus differs frompreviously observed CD4-bound states in that the typical hallmarks ofCD4-induction—such as bridging sheet formation and V3 loop exposure—areabsent or substantially reduced.

As the single-CD4-bound state could be SOSIP.664 specific, and indeedboth SOSIP.664 and DS-SOSIP.664 variant appear to be extraordinarilyrigid, DS-stabilized Envs were assessed in other contexts. When DSmutations were placed into functional virus, they ablated entry (FIG. 40). Single molecule fluorescence energy transfer (smFRET) measurements,utilizing donor-acceptors placed in the first and fourth variable Envloops of functional JR-FL viral spikes, revealed DS mutations to reducetransitions from the ground state. DS-viral spikes remained primarily inthe closed ground state, even in the presence of dodecameric CD428 (FIG.34E). Overall, the asymmetric single CD4-bound state—with fast off-ratefor CD4—appeared to be an obligatory intermediate between the unligandedstate and a more fully CD4-triggered state capable of binding multipleCD4s and co-receptor (FIG. 34F). In this context, it is noted that thehigh off-rate of CD4 in the single CD4-bound state, coupled with theslow transition to a 3:1 CD4:trimer stoichiometry, provides akinetic-based molecular mechanism for the ability of primary HIV-1isolates to resist neutralization by monomeric CD429.

Additional HIV-1 Env ectodomain variants including one or more aminoacid substitutions to stabilize the ectodomain in the prefusion matureclosed conformation are set forth in Table 13 and described herein, theantigenicity of some of which is presented in FIGS. 42-53 . Theantigenicity of exemplary protein nanoparticles including a recombinantHIV-1 Env protein is provided in FIG. 43 . The antigenicity of exemplarychimeric HIV-1 Env proteins is provided in FIG. 42 .

The unliganded Env trimer—fixed in the pre-fusion closedconformation—may be an ideal HIV-1 immunogen. We assessed DS-SOSIP.664for physical stability to conditions typically encountered duringmanufacturing and observed increased stability relative to the parentSOSIP.664 to denaturation by temperature, pH or freeze-thaw (FIG. 35A).To see if the 201C-433C substitutions might serve as a general means ofreducing CD4-induced transition in other Env antigens, the 201C-433C andSOS mutations were placed into HIV-1 Env expressed on the surface ofenzyme-treated pseudovirions (Crooks et al., J Virol, 85, 5825-5839,2011). These viral spikes were observed to resist CD4 triggering and toretain the antigenic profile of the soluble trimer for broadlyneutralizing antibodies in both BG505 and JR-FL Env backgrounds (FIG.35B). Overall, the results indicate the disulfide-shackled 201C-433Cvariants of soluble SOSIP.664 and VLP SOS to be highly desirableantigens: conformationally fixed trimers in which neutralizing epitopesare almost exclusively exposed even in the presence of CD4. It is notedthat the path to identify the 201C-433C DS substitution involved aninformation flow from broadly neutralizing antibodies, throughstructural compatibility and binding antigenicity, to obtain aconformationally fixed immunogen of appropriate antigenicity (FIG. 35C).What was unexpected was the separation of CD4 binding by the 201C-433CDS alteration into two mechanistic steps: the recognition of one CD4without any of the antigenic hallmarks of CD4 binding such as bridgingsheet formation, and the binding of more than one CD4 along withexposure or formation of characteristic CD4-induced epitopes. Inaddition to improving the antigenic specificity of unliganded HIV-1 Envimmunogens, antigenic-guided conformational fixation can thus revealadditional mechanistic steps of the HIV-1 entry pathway.

Methods

BG505 SOSIP.664 expression, purification, and deglycosylation. BG505SSOIP.664 trimer was produced in HEK 293 GnTI−/− cells via transienttransfection of the BG505 SOSIP expressing plasmid with furin andpurified as described previously (Sanders et al, PLoS pathogens 9,e1003618, 2013; Julien et al, Science, 342, 1477-1483, 2013) and inExample 1. Briefly, the BG505 SOSIP.664 expressed supernatant was passedover the 2G12 IgG-conjugated protein A column, washed withphosphate-buffered saline (PBS), and eluted with the elution buffercontaining 3M MgCl2, pH 8.5. The eluted protein was then dialyzedagainst PBS and set for deglycosylation reaction at 37° C. in thereaction buffer containing 1 mM EDTA, 150 mM NaCl, protease inhibitorcocktail (Roche), 17,000 units of Endo H/ml, and 50 mM sodium acetate,pH 5.8. The deglycosylated BG505 SOSIP was further purified withSuperdex 200 16/60 (GE Healthcare) column in the buffer containing 5 mMHEPES 7.5, 150 mM NaCl, and 0.02% NaN3. The peak corresponding totrimeric HIV-1 Env was identified, pooled and concentrated to −10 mg/mlusing an Amicon Ultra-15 centrifugal filter (MWCO 50,000, Millipore) andscreened for crystallization. For antigenicity and stability analyses,trimers were purified by affinity chromatography over a VRC01 column,purified by gel filtration over a Superdex 200 16/60 (GE Healthcare)column in buffer containing 5 mM HEPES 7.5, 150 mM NaCl, and 0.02% NaN3,and finally, passed through a 447-52D column to remove aberrant trimerspecies (FIG. 36 ).

Crystallization screening. Deglycosylated BG505 SOSIP.664 was screenedfor crystallization using 572 conditions from Hampton, Wizard andPrecipitant Synergy (Majeed et al, Structure, 11, 1061-1070, 2003)screens using a Cartesian Honeybee crystallization robot as describedpreviously (McLellan et al., Nature, 480, 336-343, 2011) and a mosquitorobot using 0.1 μl of reservoir solution and 0.1 μl of protein solution.Crystals suitable for structural determination were obtained roboticallyin 26% PEG 400, 3.2% PEG 3350, and 0.1M sodium acetate pH 5.5. Crystalswere cryoprotected in a solution containing 30% glycerol, 30% PEG 400,4% PEG 3350, and 0.1M sodium acetate pH 5.5, and flash-frozen in liquidnitrogen. Data were collected at a wavelength of 1.00 Å at the SER-CATbeamline ID-22 (Advanced Photon Source, Argonne National Laboratory).

X-ray data collection, structure solution and model building.Diffraction data were processed with the HKL2000 suite (Otwinowski &Minor, Methods Enzymol., 276, 307-326, 1997). The data were correctedfor anisotropy using the anisotropy serverservices.mbi.ucla.edu/anisoscale/ with truncations to 3.7 Å, 3.7 Λ, 3.3Å along a, b, and c axes, respectively. Structure solution was obtainedwith Phaser using 35O22- and PGT122-bound BG505 SOSIP.664 (PDB ID:4TVP10) as search models. Refinement was carried out with Phenix (Adamset al., J Synchrotron Radiat., 11, 53-55, 2004). Model building wascarried out with Coot (Emsley & Cowtan, Acta crystal. Section D, Biol.Crystal., 60, 2126-2132, 2004).

Structural analyses involving residue-specific properties. To estimatethe degree of structural flexibility in the unliganded HIV-1 trimer, wedetermined the average Cα RMSD distance for each residue position in theunliganded trimer structure (FIG. 32A). The average Cα RMSD distanceserved as a proxy for structural plasticity and was computed betweencorresponding residues after optimal superimposition onto a set of 91X-ray structures from the Protein Data Bank (PDB) (Bernstein et al. Jmol. Biol., 112, 535-542, 1977). Each domain of the unliganded trimerwas considered separately and superimposed onto the set of structuresusing the program TM-align (Zhang & Skolnick, Nucleic Acids Res., 33,2302-2309, 2005). To obtain the best possible registry betweencorresponding residues, structural superimpositions were guided by aminoacid sequence alignments when necessary. A total of 54 monomericstructures were used for superimpositions involving the gp120 domain. Togenerate FIG. 32A (left), five representative gp120 structures wereused; unliganded clade A/E HIV-1 gp120 coree (3TGT) (Kwon et al. PNAS,109, 5663-5668, 2012), b12-bound gp120 (2NY7) (Zhou et al., Nature 445,732-737, 2007), b13-bound gp120 (3IDX) (Chen et al, Science, 326,1123-1127, 2009), F105-bound gp120 (3H11)38, and CD4- and 48d-boundgp120 (3JWD) (Pancera et al. PNAS, 107, 1166-1171, 2010) structures. Forthe gp41 domain a total of 37 structures from the PDB that includedhexameric bundles as well as disordered peptides were used.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is indicativeof intrinsic amide exchange of peptide segments and is a usefultechnique to monitor dynamic characteristics of proteins in solution.Qualitative exchange profiles for observable peptides of SOSIP.664 after3s were extracted from individual HDX-MS exchange plots (Guttman et al.,Structure, 22, 974-984, 2014). The average exchange values (0-75%) weresubstituted in the B-factor field for the observed peptides of SOSIP.664coordinates and displayed within PyMol. Non-observable peptides in thedeuterium exchange experiment as well as peptides with missing electrondensity were excluded from the analysis.

Residue sequence variability was computed as the Shannon entropy foreach residue position based on a representative set of 3,943 HIV-1strains. The electrostatic potential surfaces were generated usingGRASP41.

Assessment of antibody functionality on a panel of 170 diverse HIV-1.Neutralization was measured using single-round-of-infection HIV-1Env-pseudoviruses and TZM-bl target cells, as described previously(Sarzotti-Kelsoe et al., J. Immunol. Meth., 409, 131-146, 2014).Neutralization curves were fit by nonlinear regression using a5-parameter hill slope equation. The 50% and 80% inhibitoryconcentrations (IC₅₀ and IC₈₀) were reported as the antibodyconcentrations required to inhibit infection by 50% and 80%respectively.

Computation of antibody epitope RMSD, volume overlap, and epitopepresence. HIV-1-specific antibody-antigen complex structures werecompiled from the PDB, and antibodies were defined as broadly orpoorly/non-neutralizing based on published or in-house neutralizationdata of diverse viral strains (Georgiev et al, Science, 340, 751-756,2013). Antibodies that were deemed to have insufficient evidence forbeing classified as broadly or poorly/non-neutralizing were excludedfrom the analysis. A single antibody representative was included in theanalysis in cases where multiple antibody clonal relatives were found.The epitope residues for each antibody were defined based on therespective antibody-antigen complex crystal structures, with an antigenresidue being defined as an epitope residue if any of its heavy atomswere within 5.5 Å of any antibody heavy atom. To compute the RMSDbetween the epitope residues in the antibody-antigen complex structureand the same residues in the unliganded trimer structure: (1) theepitope residues from the complex structure were aligned to theunliganded trimer structure using the align function in PyMOL, then (2)the Cα RMSD of the epitope residues was calculated. To remove outlierresidues, the top and bottom 10% of the Cα deviations were removed fromthe RMSD calculation. To calculate the volume overlap between a givenantibody and the unliganded trimer structure, the alignment from abovewas used to compute the overlap volume between the antibody from thecomplex structure and the unliganded trimer structure by using thephase_volCalc utility from Schrödinger. An antibody epitope wasconsidered as present in the unliganded trimer structure if at least 70%of the epitope residues as defined by the antibody antigen complexstructure were also present in the unliganded trimer structure. Formapping the per-residue RMSD computation onto the unliganded trimerstructure, residues part of any antibody epitope (including epitopeswith less than 70% total residues present) were included in theanalysis; if a given residue was part of more than one antibody epitope,the highest RMSD value for that residue among all epitopes was used.Antibody volume overlap values were mapped onto the unliganded trimerstructure for all residues part of the epitope for the given antibody;if a residue was part of more than one antibody epitope, then the lowestvolume overlap for that residue among all epitopes was used.Correlations of structural properties with neutralization and/or bindingdata were computed using the Spearman correlation coefficient.

Structural compatibility analysis. For a given antibody, the AntigenicStructural Compatibility (ASC) score with the HIV-1 Env unligandedpre-fusion trimer structure was computed based on a comparison to astructure of the antibody bound to an Env-derived antigen (e.g., gp120core or V3 peptide). ASC scores were computed on a 0-1 scale using thefollowing variables: (i) The fraction f of epitope residues (as definedby the structure of the antibody complex) exposed to solvent in theunliganded trimer structure was computed, with a residue consideredaccessible to solvent if its solvent-accessible surface area (SASA) wasat least half its SASA in the respective antibody complex structure; ƒwas set to 0 if less than 70% of epitope residues were present in theantigen. (ii) A resolution estimate r was used such that Cα RMSDs dbelow r=2 were not penalized in the scores. (iii) The volume overlapvalues were used to define a volume overlap factor v that is equal to 1for overlap below 200 Å3, is equal to 0 for overlap over 1000 Å3, anddecays linearly in between. Intuitively, the unliganded trimer structureis expected to be structurally compatible with an antibody if f and vare high and if the RMSD d is low, since such conditions would indicatesimilarity between the unliganded trimer structure and the Envconformation in the antibody complex. Thus, the ASC score for eachantibody with the unliganded trimer was defined by the formula: f vexp(−0.5 max(0, d−r)).

Transient transfection expression of immunogens in 96-well microplates.A 96-well microplate-formatted transient transfection expressionapproach was used to achieve high-throughput expression of variousimmunogen proteins as follows. HEK GnTi-cells were thawed and incubatedwith growth medium (293 FreeStyle Expression Medium supplemented with10% Fetal Bovine Serum and 1% streptomycin-penicillin) (Invitrogen,Calif.) at 37° C., 5% CO2, until the cells reached logarithmicphysiological growth. 24 hours prior to DNA-transient transfection, 100μl of physiologically growing cells was seeded in each well of a 96-wellmicroplate at a density of 2.5×105 cells/ml in expression medium (293FreeStyle Expression Medium supplemented with 10% Ultra-Low IgG FetalBovine Serum and 1x-Non-Essential Amino Acids) (Invitrogen, Calif.), andincubated at 37° C., 5% CO2 for 20 h. Two hours prior to transfection,100 μl of spent medium from each well was replaced with 60 μl of freshexpression medium. DNA-TrueFect-Max complexes were used fortransfection, and these were prepared by mixing 0.2 μg plasmid DNA in 10μl of Opti-MEM transfection medium (Invitrogen, Calif.) with 0.4 μl ofTrueFect-Max (United BioSystems, Md.) in 10 μl of Opti-MEM, andincubating for 15 min prior to transfection. 20 μl of the complex wasadded into each well and mixed with growing cells, and the 96-well platewas incubated at 37° C., 5% CO2. One day post transfection, 20 μl ofenriched medium (293 FreeStyle Expression Medium supplemented 25%Ultra-Low IgG Fetal Bovine Serum, 2× Non-Essential Amino Acids and 2×glutamine) was added to each well, and returned to incubator forcontinuous culture. On days three to five post transfection, the culturewas exposed to oxygen in the sterilized air once per day. After day fivepost transfection, the biological function of the expressed protein inthe supernatant in 96-well microplate was analyzed using an ELISA assay.

Antigenic analysis of stabilized HIV-1 Env trimeric immunogens in96-well microplate by antibody binding ELISA assay. The D7324antibody-coated 96-well ELISA plate was prepared by incubating 2 μg/mlof D7324 Antibody (Aalto, Ireland) in 100 μl PBS in 96 Well Flat-BottomImmuno Plate (nunc, Thermo, Ill.) overnight at 4° C., followed byremoval of coating solution and incubation of 200 μl/well, 2% (W/V) drymilk in PBS overnight at 4° C., and then the wells were washed 5 timeswith PBS+0.05% Tween 20. 30 μl of supernatant expressed in each well ofthe 96-well microplate was incubated with 70 μl of PBS in each well of aD7324 antibody-coated 96-well ELISA plate for two hours at roomtemperature (RT), and then the wells were washed 5 times with PBS+0.05%Tween 20. 100 μl of anti-specific epitope primary antibody (prepared inour lab) at a concentration of 10 μg/ml in PBS with 0.2% (W/V) dry milkand 0.2% Tween 20 was incubated into each well for 1 hour at RT, andthen the wells were washed 5 times with PBS+0.05% Tween 20. 100 μl ofHorseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody(Jackson ImmunoResearch Laboratories Inc., PA) at 1:10,000 in PBS with1.0% (W/V) dry milk and 0.2% Tween 20 was incubated into each well for30 min at RT, and then the wells were washed 5 times in PBS+0.05% Tween20. The wells were developed using TMB at RT for 10 min, and thereaction was stopped with 180 mM HCl. The readout was measured at awavelength of 450 nm. All samples were performed in duplicate.

Antigenic analysis of BG505 SOSIP.664 and mutants by MSD-ECLIA assayusing D7324 Detection. Standard 96-well bare MULTI-ARRAY Meso ScaleDiscovery (MSD) Plates (MSD, cat #L15XA-3) were coated with a panel ofHIV neutralizing and non-neutralizing monoclonal antibodies induplicates (30 μL/well) at a concentration of 10 μg/mL, diluted in 1×PBSand the plates were incubated overnight at 4° C. The following day,plates were washed (wash buffer: 0.05% Tween-20+1×PBS) and blocked with150 μL of blocking buffer [5% [W/V] MSD Blocker A (MSD, Cat #R93BA-4)]and incubated for 1 hr on a vibrational shaker (Heidolph TITRAMAX 100;CAT #P/N: 544-11200-00) at 650 rpm. All the incubations were performedat room temperature, except the coating step. During the incubation,BG505 SOSIP trimer was titrated down in a serial 2 fold dilutionsstarting at 4 μg/mL concentration of the trimer in assay diluent (1%[W/V] MSD blocker A +0.05% Tween-20). After the incubation with blockingbuffer was complete, the plates were washed and the diluted trimer wastransferred (25 μl/well) to the MSD plates and incubated for 2 hrs onthe vibrational shaker at 650 rpm. For soluble CD4 (sCD4) induction,trimer was pre-incubated with sCD4 at a constant molar concentration of1 μM for 1 hour before adding to the MSD plate. After the 2 hrincubation with trimer, the plates were washed again and secondarydetection MSD Sulfotag labeled D7324 antibody (Prior to running theassay D7324 antibody was labeled with MSD Sulfotag (MSD; Cat #R91AN-1)at a conjugation ratio of 1:15 [D7324: Sulfotag]), which was diluted inassay diluent at 5 μg/mL and was added to the plates (25 μL/well) andincubated for 1 hr on the vibrational shaker at 650 rpm. The plates werewashed and read using the 1× read buffer (MSD Read Buffer T (4×); Cat#R92TC-2) on MSD Sector Imager 2400.

Antigenic analysis of stabilized HIV-1 Env trimeric immunogens byantibody binding ELISA assay. Similar to what was done for the 96 wellplate ELISA, the D7324 (Aalto, Ireland) antibody was coated overnight at2 μg/ml in 100 μl PBS at 4° C. Wells were washed once in PBS/Tween 20(0.2%) and blocked with 200 μl/well of 2% (W/V) dry milk in PBS for onehour at room temperature (RT). The wells were then washed 5 times inPBS+0.05% Tween 20 (PBST) and purified proteins (BG505 SOSIP.664 andmutants) were then coated at either 0.5 or 2 μg/ml in PBS, 10% FBS for 2hours at RT. The wells were washed 5 times in PBS-T. 100 μl of primaryantibody at a concentration of 10 μg/ml in PBS/Tween 20 (0.2%) wasincubated into each well for 1 hour at RT, and then the wells werewashed 5 times in PBS-T. 100 μl of Horseradish peroxidase(HRP)-conjugated goat anti-human IgG antibody (Santa Cruz Biotechnology)at 1:5,000 in PBS with 0.2% Tween 20 was added to each well for 1 hourat RT. The wells were washed 5 times in PBS-T. The wells were developedusing Sureblue (KPL) at RT for 10 min, and the reaction was stopped with180 mM HCl. The readout was measured at a wavelength of 450 nm. Allsamples were performed in duplicate.

Surface plasmon resonance analysis. Affinities and kinetics of bindingto BG505 SOSIP.664 soluble trimer and its mutants were assessed bysurface plasmon resonance on a Biacore T-200 (GE Healthcare) at 20° C.with buffer HBS-EP+(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and0.05% surfactant P-20).

For assessing binding of trimer to CD41 antibody 17b and HR2-reactivepeptide C34, mouse anti-human Fc antibody was first immobilized onto twoflow cells on a CM5 chip at ˜10,000 response units (RU) with standardamine coupling protocol (GE Healthcare). Either 17b IgG or C34-Ig wasthen captured on one flow cell by flowing over a 200 nM solution at 5μl/min flow rate for two minutes. The other flow cell was used asreference. To block unliganded mouse anti-human Fc antibody, this wasfollowed by a 1-minute injection of 1 μM human Fc on both flow cells.500 nM unliganded trimer (−CD4) or a complex of 500 nM trimer+1500 nMsCD4 (+CD4) was flowed over the sample flow at a flow rate of 50 μl/minfor 2 minutes and allowed to dissociate for 5 minutes. The cells wereregenerated with two 10 μl injections of 3.0 M MgCl2, pH 7.5 at a flowrate of 100 μl/min. Blank sensorgrams were obtained by injection of thesame volume of HBS-EP+ buffer. Sensorgrams were corrected withcorresponding blank curves.

For assessing binding of trimer to sCD4, single-cycle kinetics analyseswere carried out. First, ˜2000RU of antibody 2G12 were immobilized ontwo flow cells. Next, 200 nM of trimer was injected on the sample flowcell. Finally, 5 concentrations of sCD4 (100 nM, 50 nM, 25 nM, 12.5 nM,6.25 nM) were injected incrementally in a single cycle, starting fromthe lowest concentration, followed by a dissociation phase of 30 min.Blank sensorgrams were obtained by injection of same volume of HBS-EP+buffer in place of sCD4. Sensorgrams of the concentration series werecorrected with corresponding blank curves and fitted globally withBiacore T200 evaluation software using a 1:1 Langmuir model of binding.

For assessing affinity and kinetics of antibody 17b binding, trimer wascaptured onto a 2G12 surface that was obtained by capturing 2G12 IgG ona flow cell immobilized with ˜10,000 response units (RU) of mouseanti-human Fc antibody. To block unliganded mouse anti-human Fcantibody, this was followed by a 1-minute injection of 1 μM human Fc onboth flow cells. Binding to 17b Fab was carried out in the single-cyclekinetics format with successive injections of 5 concentrations of 17bFab. Blank sensorgrams were obtained by injection of the same volume ofHBS-EP+ buffer in place of antibody Fab fragments. Sensorgrams of theconcentration series were corrected with corresponding blank curves andfitted globally with Biacore T200 evaluation software using a 1:1Langmuir model of binding.

For determining the time-course of CD4 activation of the soluble timers,17b IgG, 3074 IgG and 2G12 IgG were captured on three separate flowcells of a CM5 chip immobilized with ˜10,000 RU of mouse anti-human Fcantibody. Trimers were incubated in 4-fold molar excess of sCD4 andsamples were injected at different time-points. Blank sensorgrams wereobtained by injection of same volume of HBS-EP+ buffer in place oftrimer. To measure any change in the trimer samples on incubation,unliganded trimers were injected before and 72 hours after start of theexperiment.

Biolayer interferometry analysis. A fortéBio HTX instrument was used tomeasure affinities of BG505 SOSIP.664 and the 201C-430C variant to apanel of HIV-1 Env reactive antibodies at 30° C. All assays were carriedout with agitation set to 1,000 rpm in PBS supplemented with 1% BSA(PBS/1% BSA) using solid black 96-well plates (Geiger Bio-One). For thequaternary-specific antibodies CAP256-VRC26.09 and PGT145, the IgG (40μg/ml) was directly immobilized onto an anti-human capture sensor for300 s. Typical capture levels were between 1.2 and 1.4 nm, andvariability within a row of eight tips did not exceed 0.1 nm. Biosensortips were then equilibrated for 300 s in PBS/l % BSA buffer prior toassessment of binding to the HIV-1 trimer molecules in solution (0.015to 0.5 μM). Association was allowed to proceed for 300 s followed bydissociation for 300 s. Dissociation wells were used only once toprevent contamination. Parallel correction to subtract systematicbaseline drift was carried out by subtracting the measurements recordedfor a sensor loaded with the respiratory syncytial virus (RSV)-specificantibody D25 incubated in PBS/1% BSA.

For all other binding studies, 2G12 (60 μg/ml) was immobilized onto ananti-human capture sensor for 300 s (typical loading levels 1.0 nm)followed by incubation with either the BG505.SOSIP or the 201C-433Cvariant for 600 s (resulting in loading levels of ˜0.8 nm). The 2G12:HIV-1 Env complex was then allowed to associate with Fab molecules (2.5μM-0.2 μM) in PBS/1% BSA for 300s followed by dissociation for 300-1800s. A D25:RSV fusion glycoprotein complex was used to control fornon-specific binding of the Fab molecules. Data analysis and curvefitting were carried out using Octet software, version 8.1. Experimentaldata were fitted with the binding equations describing a 1:1interaction. Global analyses of the complete data sets assuming bindingwas reversible (full dissociation) were carried out using nonlinearleast-squares fitting allowing a single set of binding parameters to beobtained simultaneously for all concentrations used in each experiment.

Negative-stain electron microscopy. Negative-stain electron microscopysamples were diluted to about 0.03 mg/ml, adsorbed to a freshlyglow-discharged carbon-film grid for 15s, and stained with 0.7% uranylformate. Images were collected semi-automatically using SerialEM44 on aFEI Tecnai T20 with a 2 k×2 k Eagle CCD camera at a pixel size of 0.22nm/px. Particles were picked automatically and reference-free 2Dclassification was performed in EMAN245.

Differential scanning calorimetry. The heat capacity of BG505 SOSIP.664and BG505 SOSIP.664.201C-433C was measured as a function of temperatureusing a high-precision differential scanning VP-DSC microcalorimeter (GEHealthcare/Microcal, Northampton, Mass.). The samples were extensivelydialyzed against PBS, pH 7.4, and then degassed to avoid the formationof bubbles in the calorimetric cells. Thermal denaturation scans wereconducted from 10 to 100° C. at a rate of 1° C./min. The proteinconcentration was about 0.3 mg/mL.

Analytical ultracentrifugation equilibrium measurements. Analyticalultracentrifugation (AUC) equilibrium experiments were performed at 15°C., using a Beckman XL-A/I ultracentrifuge equipped with a Ti60An rotor.Data was collected using UV absorbance at 230 nm. Samples were dialyzedin Na2HPO4 10 mM, NaCl 140 mM, pH 7.4 overnight at 4° C. and loaded intosix-channel equilibrium cells with parallel sides and quartz windows.120 μL aliquots of sample diluted to 0.25 (78), 0.16 (51) and 0.087 (27)μM (μg/mL) were loaded, respectively, into three channels A, B and C ofthe cell, with three of the channels used for buffer reference. Sampleswere spun at 5000 rpm (9810*g) for 20 hours, after which four scans werecollected at a rate of 1 per hour. The rotor speed was then increased to6500 rpm (12750*g) for 10 hours, after which four additional scans werecollected at the same rate. The speed was further increased to 8000 rpm(15690*g) for another 10 hours and four more scans were recorded underthe same conditions. During the last step, the rotor speed was increasedto 10000 rpm (19620*g) for four more scans, resulting in a total of 16scans for each concentration and a total of 48 scans per protein. Thedata was processed and analyzed using HeteroAnalysis 1.1.44 software(biotech.uconn.edu/auf) and SEDPHAT46. Buffer density and protein v-barswere calculated using the SednTerp (Alliance Protein Laboratories)software. The data for all concentrations and speeds were globally fitusing nonlinear regression to an ideal monomer model. Hydrogen DeuteriumExchange (HDX). The hydrogen-deuterium exchange rates for BG505SOSIP.664 and the DS-SOSIP.664 both alone and in the presence of CD4were assessed. Complexes with soluble CD4 (D1D2) were formed byovernight incubation with a nine-fold molar excess of ligand (relativeto trimer). Proteins (10 μg) were diluted 10-fold into deuterated PBSbuffer and incubated at room temperature. Aliquots removed after 3 s, 1min, 30 min and 20 h were quenched by mixing with an equal volume ofcold 200 mM Tris-2-carboxyethyl phosphine (TCEP), 0.2% formic acid(final pH 2.5). The samples were subsequently digested with pepsin (at0.15 mg/mL) for 5 min on ice, flash frozen in liquid nitrogen, andstored at ˜80° C. For LC-MS analysis, samples were thawed on ice for 5minutes and manually injected onto a Waters BEH 1.7 μm 1.2×5 mm trapcolumn (Waters) flowing 0.1% TFA at 200 μL/min. After 3 minutes ofwashing the peptides were resolved over a Hypersil 1×50 mm 2.1 μm C18column (Thermo Scientific) using a gradient of 15 to 40% B in 8 minutes(A: 0.05% TFA 5% ACN; B: 0.05% TFA 80% ACN). Eluted peptides wereanalyzed with a Waters Synapt Q-TOF mass spectrometer. Peptideidentification and exchange analysis were as described previously(Guttman et al, Structure, 22, 974-984, 2014).

Neutralization of viral entry. The point mutations were introduced intofull-length Env clone BG505.W6M.C212 in expression vectorpcDNA3.1/V5-His-TOPO (Invitrogen). Pseudotyped, single round of entryvirus was produced as described in Shu et at (Shu et al., Vaccine, 25,1398-1408, 2007). Briefly, plasmid DNA was used to transfect 293T cellsalong with an envelope-deficient HIV-1 subtype A proviral plasmid,SG3dEnv48 to generate pseudotyped viral particles. Serial dilutions ofthe pseudovirus stocks were added to TZMbl reporter cells, and two dayslater the activity of the luciferase reporter gene in infected cells wasassessed with a Luciferase Assay kit (Promega) and measured in aluminometer; activity was reported as Relative Light Units (RLU).

smFRET on JR-FL viral spikes. Briefly, HEK293 cells were transfected ata 40:1 ratio of wild-type HIV-1JR-FL or HIV-1JR-FL 201C-433C Env todually V1-Q3/V4-A1 tagged Env and the additional presence of pNL4-3 AenvART. Virus was concentrated from supernatants 40 h post-transfection anddually labelled overnight in a reaction with 0.5 μM Cy3B(3S)-cadaverine,0.5 μM Cy5(4S)COT-CoA, 0.65 μM transglutaminase (Sigma), and 5 μM AcpSat room temperature. After addition of DSPE-PEG2,000-biotin (Avanti) at0.02 mg/ml (30 min), the viruses were purified on a 6-18% Optiprepgradient in 50 mM Tris pH 7.4, 100 mM NaCl and stored at −80° C. ForsmFRET imaging, viruses were immobilized on streptavidincoated quartzmicrofluidic devices and imaged at room temperature on a wide-fieldprism-based TIRF instrument equipped with an Opus 532 nm laser (LaserQuantum). Donor and acceptor fluorescence were collected through a1.27-NA 60× water-immersion objective (Nikon), and recorded using anORCA-Flash4.0 sCMOS camera (Hamamatsu) at 25 frames/s for 80 s. AllsmFRET imaging experiments were performed in buffer containing 50 mMTris pH7.5, 100 mM NaCl, and a cocktail of triplet-state quenchers andoxygen scavengers. The conformational effects of dodecameric sCD4D1D2(sCD4D1D2-Igatp) on wild-type and HIV-1JR-FL 201C-433C mutant Env weretested after incubation with the ligand for 30 min at 0.01 mg/ml.Histograms were fitted into three-state Gaussian curves; and occupanciesof each FRET state were calculated from histogram fitting. FollowingHidden Markov Modeling, all transitions were displayed in transitiondensity plots (TDP).

Assessment of physical stability. To assess the physical stability ofthe closed, prefusion conformation of trimeric BG505 SOSIP and 201C-433Cproteins, the proteins were subjected to a variety of pharmaceuticallyrelevant stresses such as extreme pH, high temperature, low and highosmolarity, as well as repeated freeze/thaw cycles. The physicalstability of treated BG505 SOSIP and 201C-433C proteins was evaluated bymeasuring the retention of binding to the quaternary-specificV1V2-directed antibodies CAP256-VRC26.09 and PGT145 and induction ofbinding to the V3-loop antibody 447.52D and the CD41 antibody 17b whichis not observed in the closed prefusion conformation of the SOSIPtrimer. The retention of binding to CD4-Ig and VRC01 was also measured.In the pH treatment experiments, HIV-1 proteins were prepared at aninitial concentration of 125 μg/ml, and pH was adjusted using either pH3.5 or pH 10 with 0.5 M citrate, pH 2.8 or 1 M CAPS, pH 10.5 bufferrespectively and incubated at room temperature for 60 minutes beforereturning the pH to pH 7.5 using 1 M Tris, pH 8.5 or 1 M Tris, pH 7.0,respectively. In the temperature treatment experiments, HIV-1 proteinsat 125 μg/ml concentration were incubated at 50° C., 70° C. and 90° C.for 60 minutes in PCR cyclers with heated lids to prevent evaporationand ramp rates of 2.5° C./s. To assess antigenic characteristicsfollowing extremes of low and high osmolarity, spin desalting columns(Thermo Scientific) were used to buffer exchange the proteins intoeither 5 mM Tris, pH 7.5, 10 mM NaCl or 3 M MgCl2, pH 7.5. Proteins wereincubated at room temperature for 60 minutes before buffer exchange into1×PBS, pH 7.4 and concentrating the sample to 125 μg/ml concentration.The freeze/thaw treatment was carried out by repeatedly flash freezingprotein in liquid nitrogen and thawing at 37° C. ten times. All proteinsolutions were supplemented with 0.2% BSA for a final HIV-1 proteinconcentration of 100 μg/ml and antibody binding measurements werecarried out using a fortéBio Octet HTX instrument. Assays were performedat 30° C. with agitation set to 1000 rpm in tilted black 384-well plates(Geiger Bio-One) and a volume of 50 μl/well. Anti-human Fc probes wereloaded with full-length IgG of the antibodies mentioned above at 50μg/ml in PBS buffer for 180 seconds, which were then equilibrated for240 seconds in PBS+0.2% BSA before being used to immobilize treated oruntreated BG505 SOSIP and 201C-433C proteins for 300 seconds. Parallelmeasurements of antibody binding to PBS+0.2% BSA and soluble recombinantInfluenza hemagglutinin in PBS+0.2% BSA were used to assess systematicbaseline drift and non-specific binding. The fractional degree ofretention of quaternary structure integrity is reported as the ratio ofsteady state binding level before and after stress treatment. To assessthe physical stability of the closed, prefusion conformation of trimericBG505 SOSIP, A433P and 201C-433C proteins over time, we incubated a 40nM solution of each trimer in HBS-EP+buffer at 4 differenttemperatures-4° C., 20° C., 37° C. and 42° C. Aliquots were taken atdifferent time points over the course of 10 days, and retention ofquaternary structure in the trimers was assessed by SPR by flowing overantibody VRC26.09 captured on an Fc surface. A parallel lane with 2G12captured on it served as control for equal protein loading. Blanksubtractions were carried out as described in the SPR section above. Thefractional degree of retention of quaternary structure integrity isreported as the ratio of steady state binding level before and afterincubation. Virus-Like Particles ELISAs. ELISAs were performed asdescribed previously 49. Briefly, Immulon II plates were coatedovernight at 4° C. with VLPs at 20 times their concentration intransfection supernatants. Wells were washed with PBS and then blockedwith 4% bovine serum albumin/10% fetal bovine serum in PBS. Variousbiotinylated monoclonal antibodies (biotinylated using sulfo-NHSXbiotin,Thermo), and CD4-IgG2 were then titrated in the presence or absence of afixed concentration of 2 μg/ml soluble CD4. Alkaline phosphataseconjugated to streptavidin (Vector Laboratories, Burlingame, Calif.; todetect biotinylated mAbs) or anti-Fc (Accurate, Westbury, N.Y.; todetect CD4-IgG2) and SigmaFAST p-nitrophenyl phosphate tablets (Sigma)were then used to detect binding. Plates were read at 405 nm.

Figures. Structure figures were prepared using PYMOL50.

Coordinates. The atomic coordinates of an asymmetric unit of the crystalstructure of the unliganded trimeric HIV-1 Env ectodomain in theprefusion mature closed conformation are recited in Table 3 submitted asan ASCII text named “Table_3.txt” (˜0.7 MB, created on Aug. 7, 2014) inU.S. Provisional Application No. 62/046,059, filed Sep. 4, 2014, andhave been deposited with the Protein Data Bank as Acc. No. 47MJ. Theatomic coordinates of the crystal structure of an unliganded trimericHIV-1 Env ectodomain in the prefusion mature closed conformation arerecited in Table 4 submitted as an ASCII text named “Table_4.txt” (˜2MB, created on Aug. 7, 2014) in U.S. Provisional Application No.62/046,059, filed Sep. 4, 2014.

Example 3 Production of IV-1 Env Protein Covalently Linked to Antibody

This example provides an exemplary protocol for producing a recombinantHIV-1 Env protein covalently linked to a broadly neutralizing antibody.

HIV-1 Env expression construct were designed to contain a cysteinemutation at a key complementary site at the antibody complex interfaceand a protease cleavable Streptactin II tag C-terminal of the HIV-1 Envresidue 664. The respective antibodies were mutated to contain acysteine at a key complementary site at the antibody complex interfaceand the antibody heavy chain had a C-terminal cleavable His6 tag afterthe Fab region. In the case of covalently linking VRC01 to HIV-1 gp140trimer, mutations 459C in HIV-1 Env and 60C in VRC01 heavy chain enabledcovalent assembly of the complex within the producer cells.

DNA for the HIV-1 Env, antibody heavy, antibody light and furin weremixed together in a molar ratio of approximately 1:0.25:0.25:0.5 andtransfected into suspension HEK 293F cells. 7 days post transfection thesupernatants were harvested, clarified and filtered. The media waspassed through NiNTA resin and after washing with PBS eluted in 250 mMimidazole. The eluate was passed over a 2 ml streptactin column, washedwith 5 ml wash buffer and eluted in 3 ml elution buffer using themanufacturer's buffer formulations. The resulting eluate wasconcentrated to 1.5 ml and passed through a Superdex S200 gel filtrationcolumn equilibrated in phosphate buffered saline and the main peakcontaining the covalently linked complex was verified to contain theintermolecular disulfide bond by reducing and non-reducing SDS-PAGE.Fractions corresponding to the covalently linked antibody-Env complexwere pooled and flash frozen in liquid nitrogen and stored at −80 C.

Example 4 Single Chain HIV-1 Env Proteins

HIV-1-Env constructs from various strains were synthesized and includethe “SOS” mutations (A501C, T605C), the isoleucine to proline mutationat residue 559 (1559P), and the glycan site at residue 332 (T332N);mutating the gp120/gp41 cleavage site to a ten-amino-acid linker; andtruncating the C terminus to residue 664 (all HIV-1 Env numberingaccording to the HXB2 nomenclature). This construct in the case of theBG505 strain is referred to as bC101n.

The bC101n construct was transfected in HEK 293 F cells using 1 mgplasmid DNA and transfection supernatants were harvested after 7 days,and passed over either a 2G12 antibody- or VRC01 antibody-affinitycolumn. After washing with PBS, bound proteins were eluted with 3MMgCl₂, 10 mM Tris pH 8.0. The eluate was concentrated to less than 5 mlusing a Centricon-70 and applied to a Superdex 200 column, equilibratedin phosphate buffered saline. The peak corresponding to trimeric HIV-1Env was identified, pooled, and concentrated or flash-frozen in liquidnitrogen and stored at −80° C.

Constructs such as bC101n were also designed with a C-terminal Thrombincleavage site, His₆-tag, Streptactin II tag to enable purification asdescribed in McLellan et al. Science 340, 1113-1117, 2013. Briefly,supernatants were harvested after 7 days, and passed over NiNTA affinitycolumn. After washing with PBS, bound proteins were eluted with 250 mMimidazole. The eluate was concentrated to less than 3 ml using aCentricon-70 and applied to a 2 ml Streptactin column, equilibrated inphosphate buffered saline. The sample was eluted in 8 ml of elutionbuffer. The eluate was concentrated to less than 5 ml using aCentricon-70 and applied to a Superdex 200 column, equilibrated inphosphate buffered saline. The peak corresponding to trimeric HWV-1 Envwas identified, pooled, and concentrated or flash-frozen in liquidnitrogen and stored at −80° C.

Example 5 Chimeric HIV-1 Env Proteins

This example describes the design and production of chimeric HIV-1 Envimmunogens based on diverse HWV-1 strains. In the context of inducing animmune response in a subject that can control infection across multipleHIV-1 strains, the use of immunogens based on diverse HIV-1 strains canovercome the intrinsic sequence diversity of HIV-1 Env.

The structural data provided in the prior Examples illustrates that theHIV-1 Env ectodomain in the prefusion mature closed conformationincludes a “base” or “platform” including three gp41 molecules, each ofwhich wrap their hydrophobic core around the extended N- andC-termini-strands of gp120 (see, e.g., FIGS. 45-46 ). Accordingly,chimeric HIV-1 Env ectodomains were designed with N- and C-terminalregions of gp120 and the gp41 ectodomain from a first HIV-1 strain, andthe remainder of gp120 from a second HIV-1 strain. As illustrated inFIGS. 45-46 , these chimeric HIV-1 Env proteins allow for a gp41“platform” on which a chimeric gp120 sequence can be presented to theimmune system. The variable gp120 molecule sits on top of a gp41 fromthe BG505 strain (with SOSIP substitutions), with the N- and C-terminalregions of gp120 also from the BG505 strain. As shown in red in FIG. 47, the N- and C-terminal regions of gp120 can include all or part of theβ-4 strand, the β-3 strand, the β26 strand, the β25 strand and/or the α5helix.

The interface between the gp41 and gp120 proteins in the HIV-1 Envtrimer includes gp120 residues 46-54, 70-75, 84-89, 99, 102, 106, 107,114, 215, 220-224, 226, 244, 471-473, and 476-477 (“Interface Residueset A”). In FIG. 47 , these residues are shaded light blue. Accordingly,additional chimeric HIV-1 Env ectodomains were designed with N- andC-terminal regions of gp120, Interface Residue Set A, and the gp41ectodomain from a first HIV-1 strain, and the remainder of gp120 from asecond HIV-1 strain (these chimeras include reference to “InterfaceResidue Set A” in columns 6 or 7 of the tables in Table 13). Thesechimeric HIV-1 Env ectodomains include an expanded “platform” on which achimeric gp120 sequence can be presented to the immune system.

A chimeric HIV-1-Env construct was synthesized that includes gp120residues 31-45 and 478-507, and gp41 residues 512-664 from the BG505strain with SOSIP and 332N substitutions (e.g., as set forth as SEQ IDNO: 3), and the remainder of the gp120 residues (46-477) from the3301_V1_C24 HIV-1 strain (SEQ ID NO: 751), which is a clade C virus. Theprotease cleavage site separating gp120/gp41 was mutated to include sixarginine residues, and the C-terminus of gp41 was set at position 664(all HIV-1 Env numbering according to the HXB2 nomenclature). The aminoacid sequence of the resulting chimeric Env protein is provided as SEQID NO: 384 (3301_V1_C24_bg505-NCgp120+gp41.SOSIP). Additional variantswere designed and produced, including a chimeric HIV-1 Env ectodomaintrimer having gp41, and gp120 N- and C-terminal region sequences fromthe BG505 strain (with SOSIP substitutions), with the remaining gp120sequence from the ZM53, or 25925-2.22 strains. The correspondingchimeric proteins were termed ZM53_BG505-NCgp120_gp41.SOSIP (SEQ ID NO:386), 25925-2.22_BG505-NCgp120_gp41.SOSIP (SEQ ID NO: 383), and3301_V1_C24_BG505-NCgp120+gp41.SOSIP (SEQ ID NO: 384). Expression andpurification was performed as described in Examples 1 and 2 above.

A further variant was produced, SEQ ID NO: 382(CNE58_SU-strandC_bg505-NCgp120+gp41.SOSIP) that includes gp41 and gp120N- and C-terminal regions (31-45 and 478-507, respectively) fromBG505.SOSIP.664, with residues 166-173 (V1V2 strand C) from CAP256 SU,and the rest of gp120 from the CNE58 strain.

DNA constructs encoding the chimeric Env proteins were transfected inHEK 293 F cells using 1 mg plasmid DNA and 250 μg plasmid encoding Furinas described previously (Sanders, PLoS pathogens 9, e1003618, 2013,incorporated by reference herein). Transfection supernatants wereharvested after 7 days, and constructs that ended at residue 664 werepurified using a GNA-Lectin affinity column as described in Pejchal etal, Science, 334: 1097-1103, 2011, incorporated by reference herein.Briefly, supernatants were passed over the GNA-lectin-affinity columnand after washing with PBS, bound proteins were eluted with 1 M methylα-D-mannoside. The eluate was concentrated to less than 5 ml using aCentricon-70 and applied to a Superdex 200 column, equilibrated inphosphate buffered saline. The peak corresponding to trimeric HIV-1 Envwas identified, pooled, and concentrated or flash-frozen in liquidnitrogen and stored at −80° C.

Constructs were also designed with a C-terminal Thrombin cleavage site,His₆-tag, Streptactin II tag to enable purification as described inMcLellan et al. Science 340, 1113-1117, 2013. Briefly, supernatants wereharvested after 7 days, and passed over NiNTA affinity column. Afterwashing with PBS, bound proteins were eluted with 250 mM imidazole. Theeluate was concentrated to less than 3 ml using a Centricon-70 andapplied to a 2 ml Streptactin column, equilibrated in phosphate bufferedsaline. The sample was eluted in 8 ml of elution buffer. The eluate wasconcentrated to less than 5 ml using a Centricon-70 and applied to aSuperdex 200 column, equilibrated in phosphate buffered saline. The peakcorresponding to trimeric HIV-1 Env was identified, pooled, andconcentrated or flash-frozen in liquid nitrogen and stored at −80° C.

The 3301_V1_C24_bg505-NCgp120+gp41.SOSIP andZM53_BG505-NCgp120_gp41.SOSIP chimeras had nearly full gp120/gp41cleavage, as shown by SDS-page (FIG. 48 , left). Further, these chimeraseluted from the purification column in trimeric form under a single peak(FIG. 48 , right). The antigenicity of the3301_V1_C24_bg505-NCgp120+gp41.SOSIP, ZM53_BG505-NCgp120_gp41.SOSIP,25925-2.22_BG505-NCgp120_gp41.SOSIP, and3301_V1_C24_BG505-NCgp120+gp41.SOSIP chimeric HIV-1 Env trimers wasinterrogated using Meso Scale Discovery multi-arrayelectro-chemiluminescence (FIG. 49 ). As illustrated in FIG. 49 , thechimeras specifically bound to quaternary specific antibodies, but notto non- or poorly-neutralizing antibodies.

Additional variants were designed and produced, including a chimericHIV-1 Env including a BG505 gp120 sequence with SOSIP substitutions, anda CAP45 gp41 sequence. The sequence of this chimera is provided as SEQID NO: 772. Structural analysis of the gp120 and gp41 contacts confirmthat there is minimal disruption between the gp120-gp41 interface whensubstituting this strain. The BG505.SOSIP/CAP45 chimera had nearly fullgp120/gp41 cleavage, as shown by SDS-page (FIG. 50 , left). Further,antigenic analysis by ELISA confirmed that this chimera specificallybound to quaternary specific antibodies, but not to non- orpoorly-neutralizing antibodies (FIG. 50 , right).

The neutralization profile of several neutralizing and non-neutralizingantibodies was compared with the antigenic profile of a chimeric HIV-1Env ectodomain based on the native DU156 virus. As shown in FIG. 51 ,the neutralization profile correlates with the antigenic profile,particularly for the chimeric HIV-1 Env ectodomain including the201C/433C substitutions.

Many additional chimeric HIV-1 Env ectodomain proteins were designed andproduced, including those provided as SEQ ID NOs: 379-386, 579-595,764-772, 856-1056, 1077-1098, and 1114-1200. The details of the designof each of these chimeric Env proteins are provided in Table 13.Additional recombinant HIV-1 Env ectodomains including stabilizingsubstitutions and based on more HIV-1 strains were also produced,including those provided as SEQ ID NOs: 1057-1077. The recombinant HIV-1Env ectodomains were expressed in cells and the corresponding antigeniccharacteristics of each ectodomain was evaluated by bind antibodybinding assay.

Binding to several different antibodies was assayed to evaluate theantigenic profile of each the recombinant HIV-1 Env proteins (FIGS. 44and 53 ). The antibodies tested included VRC26 and PGT145 (which bindV1V2 specific epitopes present on the prefusion mature closedconformation of HIV-1 Env), F105 (which binds an epitope that is notpresent on the prefusion mature closed conformation of HIV-1 Env), 17b(which binds a CD4-induced epitope) in the presence or absence of sCD4,PGT151 and 35O22 (which bind conformational epitopes including gp120 andgp41 residues of HIV-1 Env in its prefusion mature conformation), PGT122(which binds a conformation epitope including V1V2 and V3-glycanresidues), 447-52D (which binds a V3-loop epitope), and VRC01 (whichbinds the CD4 binding site).

FIG. 53 shows the antigenic readout of many chimeric HIV-1 Envectodomains, each of which was stabilized in a prefusion matureconformation using the SOSIP substitutions and a 201C-433C disulfidebond. The antigenicity assays show that all of the recombinant HIV-1 Envproteins tested exhibited little to no binding to the 17b antibody, evenin the presence of a molar excess of soluble CD4. This findingillustrates the effectiveness of this mutation (201C-433C) forstabilizing the HIV-1 Env ectodomain in a conformation that is resistantto CD4-induced change.

Additionally, bioinformatics algorithms were used to identify chimericHIV-1 Env ectodomains that exhibited relatively strong binding toquaternary-specific antibodies (e.g., VRC26) and relatively weak bindingto weakly neutralizing antibodies (e.g., F105). Based on thesealgorithms, several chimeras of particular interest were identified,including the following:

DU422.01-chim_d7324.201C-433C (SEQ ID NO: 964)

ZM106.9-chim_d7324.201C-433C (SEQ ID NO: 1025)

CH038.12-chim_d7324.201C-433C (SEQ ID NO: 938)

16055-2.3-chim_d7324.201C-433C (SEQ ID NO: 872)

ZM55.28a-chim_d7324.201C-433C (SEQ ID NO: 1098)

CH117.4-chim_d7324.201C-433C (SEQ ID NO: 940)

ZM53.12-chim_d7324.201C-433C (SEQ ID NO: 1034)

25925-2.22-chim_d7324.201C-433C (SEQ ID NO: 881)

B1369.9A-chim_d7324.201C-433C (SEQ ID NO: 924)

3301.V1.C24-chim_d7324.201C-433C (SEQ ID NO: 888)

CAP45.G3-chim_d7324.201C-433C (SEQ ID NO: 937)

C1080.c3-chim_d7324.201C-433C (SEQ ID NO: 930)

286.36-chim_d7324.201C-433C (SEQ ID NO: 856)

MW965.26-chim_d7324.201C-433C (SEQ ID NO: 978)

CNE55-chim_d7324.201C-433C (SEQ ID NO: 953)

C4118.09-chim_d7324.201C-433C (SEQ ID NO: 933)

DU156.12-chim_d7324.201C-433C (SEQ ID NO: 962)

TH966.8-chim_d7324.201C-433C (SEQ ID NO: 1010)

6545.V4.C1-chim_d7324.201C-433C (SEQ ID NO: 908)

620345.cl-chim_d7324.201C-433C (SEQ ID NO: 902)

0921.V2.C14-chim_d7324.201C-433C (SEQ ID NO: 871)

AC10.29-chim_d7324.201C-433C (SEQ ID NO: 917)

QH209.14M.A2-chim_d7324.201C-433C (SEQ ID NO: 990)

50 MB201.A1-chim_d7324.201C-433C (SEQ ID NO: 973)

These chimeras include gp120 sequences from several different HIV-1subtypes, including subtype A (BI369.9A, MB201.A1, QH209.14M.A2),subtype B (AC10.29), subtype C (0921.V2.C14, 16055-2.3, 25925-2.22,286.36, CAP45.G3, DU156.12, DU422.01, MW965.26, ZM53.12, ZM55.28a,ZM106.9), subtype CRF AC (3301.V1.C24, 6545.V4.C1), subtype CFR AE(620345.c1, C1080.c3, C4118.09, CNE55, TH966.8) and subtype CRF BC(CH038.12, CH117.4). Thus, these results demonstrate that the strategiesfor stabilizing chimeric HIV-1 Env ectodomains in the prefusion matureclosed conformation disclosed herein can be applied across a diversearray of HIV-1 strains.

Based on the antigenic characteristics of the assayed chimeras,additional chimeric HIV-1 Env ectodomains were constructed. By comparingthe sequences for assayed chimeras with good antigenic characteristics(e.g., strong binding to VRC26 and low binding to F105) to chimeras withpoor antigenic characteristics (e.g., low binding to VRC26 and strongbinding to F105), residue positions within gp120 that had differentamino acid composition in the former vs. the latter set of chimeras wereidentified using bioinformatics algorithms. In a non-limitingembodiment, such residue positions included Residue Set B (SEQ1114-1142): 133-134, 164, 169, 308, and 316 from BG505. In anothernon-limiting embodiment, such residue positions included the expandedset Residue Set C (SEQ 1143-1171: 49, 133-134, 149-152, 164,169, 188,190, 211, 223, 252, 281, 293, 308, 316, 336, 340, 352,360,362-363,369,372, 393, 410, 432, 442, 444, 446, 474, and 476 fromBG505. In another non-limiting embodiment, such residue positionsincluded the expanded set Residue Set C+Residue Set D (SEQ 1172-1200):46, 60, 62-63, 84-85, 87, 99, 102, 130, 132, 135, 153, 158, 160-161,165-167, 171-173, 175, 177-178, 181, 184-185, 189, 202, 232, 234, 236,240, 268-271, 275, 277, 287, 289, 292, 295, 297, 305, 315, 317, 319,322, 328, 330, 332-335, 337, 339, 343-347, 350-351, 357, 371, 375, 379,387, 389, 394, 411, 412-413, 415, 424, 426, 429, 440, 460-461, 465, 475,and 477 from BG505.

Example 6 Protein Nanoparticles Including Recombinant HIV-1 Env Proteins

This example provides an exemplary protocol for producing a proteinnanoparticle including a recombinant HIV-1 Env protein that isstabilized in a prefusion mature conformation.

BG505 SOSIP.664 linked to nanoparticles (e.g. Ferritin) wascotransfected with furin in HEK 293 S GnTI−/− cells using 500 μgsplasmid DNA and 125 μgs of furin. Transfection supernatants wereharvested after 7 days, and passed over either a 2G12 antibody- or VRC01antibody-affinity column. After washing with PBS, bound proteins wereeluted with 3M MgCl₂, 10 mM Tris pH 8.0. The eluate was concentrated toless than 5 ml with Centricon-70 and applied to a Superdex 200 column,equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% azide. The peakcorresponding to the nanoparticle size was identified, pooled, andconcentrated or flash-frozen in liquid nitrogen and stored at −80° C.

Alternatively, other methods as described in Kanekiyo et al., Nature,499, 102-106, 2013, incorporated by reference herein, can be used topurify nanoparticles including recombinant HIV-1 Env proteins.

Example 7 Immunization of Animals

This example describes exemplary procedures for the immunization ofanimals with the disclosed immunogens, and measurement of thecorresponding immune response.

In some examples nucleic acid molecules encoding the disclosedimmunogens are cloned into expression vector CMV/R. Expression vectorsare then transfected into 293F cells using 293Fectin (Invitrogen,Carlsbad, Calif.). Seven days after transfection, cell culturesupernatant is harvested and passed over either a 2G12 antibody- orVRC01 antibody-affinity column. After washing with PBS, bound proteinswere eluted with 3M MgCl₂, 10 mM Tris pH 8.0. The eluate wasconcentrated to less than 5 ml with Centricon-70 and applied to aSuperdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl,0.02% azide. The peak corresponding to trimeric HIV-1 Env wasidentified, pooled, and concentrated or flash-frozen in liquid nitrogenand stored at −80° C. Some proteins are purified using HiTrap IMAC HPColumn (GE, Piscataway, N.J.), and subsequent gel-filtration usingSUPERDEX™ 200 (GE). In some examples the 6× His tag is cleaved off using3C protease (Novagen, Madison, Wis.). For vaccinations with thedisclosed immunogens 4-6 months old guinea pigs (Strain Hartley)(CharlesRiver Laboratories, Mass.) are immunized using polyIC (High molecularweight, InvivoGen Inc, CA) as the adjuvant. Specifically, four guineapigs in each group are vaccinated with 25 μg of protein and 100 μg ofpolyIC in 400 μl intramuscularly (both legs, 200 μl each leg) forexample at week 0, 4, 8, 12, 22. Sera are collected for example at week2 (Post-1), 6 (Post-2), 10 (Post-3), 14 (Post-4) and 24 (Post-5), andsubsequently analyzed for their neutralization activities against apanel of HIV-1 strains, and the profile of antibodies that mediate theneutralization. The immunogens are also used to probe for guinea piganti-sera for existence of HIV-1 neutralizing antibodies in theanti-sera, such as antibodies that compete for binding to therecombinant HIV-1 Env ectodomain trimer with PGT122, PGT145, PGT151,and/or VRC26.

Example 8 Immunization of Non-Human Primates

This example describes exemplary procedures for the immunization ofnon-human primates with the disclosed immunogens, and measurement of thecorresponding immune response.

In some examples nucleic acid molecules encoding the disclosedimmunogens are cloned into expression vector CMV/R. Expression vectorsare then transfected into 293F cells using 293Fectin (Invitrogen,Carlsbad, Calif.). Seven days after transfection, cell culturesupernatant is harvested and passed over either a 2G12 antibody- orVRC01 antibody-affinity column. After washing with PBS, bound proteinswere eluted with 3M MgCl₂, 10 mM Tris pH 8.0. The eluate wasconcentrated to less than 5 ml with Centricon-70 and applied to aSuperdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl,0.02% azide. The peak corresponding to trimeric HIV-1 Env wasidentified, pooled, and concentrated or flash-frozen in liquid nitrogenand stored at −80° C. Some proteins are purified using HiTrap IMAC HPColumn (GE, Piscataway, N.J.), and subsequent gel-filtration usingSUPERDEX™ 200 (GE). In some examples the 6× His tag is cleaved off using3C protease (Novagen, Madison, Wis.). For vaccinations with thedisclosed immunogens, Indian origin Rhesus Macaque (bodyweights morethan 2 kg) are immunized with polyIC-LC as the adjuvant. Specifically,five monkeys in each group are vaccinated with 100 μg of protein and 500μg polyIC-LC in 1 ml intramuscularly in the Quadriceps muscle forexample at week 0, 4, 20. Sera are collected for example at week 2(Post-1), 6 (Post-2), 24 (Post-3), and subsequently analyzed for theirneutralization activities against a panel of HIV-1 strains, and theprofile of antibodies that mediate the neutralization. The immunogensare also used to probe for Rhesus Macaque anti-sera for existence ofHIV-1 neutralizing antibodies in the anti-sera, such as antibodies thatcompete for binding to the recombinant HIV-1 Env ectodomain trimer withPGT122, PGT145, PGT151, and/or VRC26.

Example 9 Assaying Serum Neutralization Activity

Following immunization with a disclosed immunogen (e.g., as describedabove) serum can be collected at appropriate time points, frozen, andstored for neutralization testing. The serum neutralization activity canbe assayed using various assays, such as a pseudoviruses neutralizationassay.

In some embodiments, the serum neutralization activity can be assayedessentially as previously described (see, e.g., Georgiev et al.,Science, 340, 751-756, 2013, which is incorporated by reference hereinin its entirety). Briefly, frozen serum from the immunized subject isheat-inactivated at 56° C. for 30 min prior to the assay. Pseudovirusstocks are prepared by co-transfection of 293T cells with an HIV-1Env-deficient backbone and an expression plasmid for the Env gene ofinterest. The serum to be assayed is diluted in Dulbecco's modifiedEagle medium-10% FCS (Gibco) and mixed with pseudovirus. After 30 min,10,000 TZM-bl cells are added, and the plates are incubated for 48hours. Assays are developed with a luciferase assay system (Promega,Madison, Wis.), and the relative light units (RLU) are read on aluminometer (Perkin-Elmer, Waltham, Mass.). The percent neutralizationis calculated as follows: % neutralization=100×(V₀−V_(n))/V_(o), whereV_(n) is the RLU in the virus and antibody wells and V₀ is the RLU inthe virus-only wells. The reciprocal dilution at which 50% of the virusis neutralized (ID50) is computed for each virus-serum pair. To accountfor background, a cutoff of ID50>=40 is used as a criterion for thepresence of serum neutralization activity against a given virus.

Standard panels of Env proteins from selected HIV-1 strains have beendeveloped for co-expression with the Env-deficient backbone in theneutralization assay (see, e.g., Georgiev et al., Science, 340, 751-756,2013, incorporated by reference herein). For example, the standard panelcan include Env proteins from HIV-1 strains from Clade A (KER2018.11,Q23.17, Q168.a2, Q769.h5, and RW020.2), Clade B (BaL.01, 6101.10,BG1168.01, CAAN.A2, JR-FL, JR-CSF.JB, PVO.4, THRO4156.18, TRJO4551.58,TRO.11, and YU2), and Clade C (DU156.12, DU422.01, ZA012.29, ZM55.28a,and ZM106.9). An additional standard panel is provided in Table S5 ofGeorgiev et al. (Science, 340, 751-756, 2013, which is incorporated byreference herein in its entirety) and Table 1 of Seaman et al., J.Virol., 84, 1439-1452, 2005, which is incorporated by reference hereinin its entirety).

Example 10 Treatment of Subjects

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from HIV-1 that can be treatedby eliciting an immune response, such as a neutralizing antibodyresponse to HIV-1. In particular examples, the method includes screeninga subject having, thought to have or at risk of having a HIV-1infection. Subjects of an unknown infection status can be examined todetermine if they have an infection, for example using serologicaltests, physical examination, enzyme-linked immunosorbent assay (ELISA),radiological screening or other diagnostic technique known to those ofskill in the art. In some examples, subjects are screened to identify aHIV-1 infection, with a serological test, or with a nucleic acid probespecific for a HIV-1. Subjects found to (or known to) have a HIV-1infection can be administered a disclosed immunogen (such as arecombinant HIV-1 Env stabilized in a prefusion mature closedconformation) that can elicit an antibody response to HIV. Subjects mayalso be selected who are at risk of developing HIV, such as subjectsexposed to HIV.

Subjects selected for treatment can be administered a therapeutic amountof a disclosed immunogen as disclosed herein. For example, a disclosedHIV-1 Env protein stabilized in a prefusion mature closed conformationcan be administered at doses of 0.5 μg/kg body weight to about 1 mg/kgbody weight per dose, such as 1 μg/kg body weight-100 μg/kg body weightper dose, 100 μg/kg body weight-500 μg/kg body weight per dose, or 500μg/kg body weight-1000 μg/kg body weight per dose. However, theparticular dose can be determined by a skilled clinician. The immunogencan be administered in one or several doses, for example in aprime-boost vaccination. The mode of administration can be any used inthe art. The amount of agent administered to the subject can bedetermined by a clinician, and may depend on the particular subjecttreated. Specific exemplary amounts are provided herein (but thedisclosure is not limited to such doses).

Example 11 Inducing a Neutralizing Immune Response in an Animal Model

This example provides data showing that a HIV-1 Env ectodomain timerstabilized in the prefusion mature conformation can induce aneutralizing immune response in multiple animal models.

Two months old Hartley guinea pigs (four animals per study group) or NewZealand white rabbits (five animals per study group) were immunized atweeks 0, 4, and 16, as follows: Guinea pigs

1) BG505 SOSIP (25 μg) and polyIC (100 μg) as adjuvant

2) BG505 SOSIP DS (25 μg) and polyIC (100 μg) as adjuvant

3) BG505 SOSIP (25 μg) and Matrix M (25 μg) as adjuvant

4) BG505 SOSIP DS (25 μg) and Matrix M (25 μg) as adjuvant Rabbits

1) BG505 SOSIP (30 μg) and Matrix M (30 μg) as adjuvant

2) BG505 SOSIP DS (30 μg) and Matrix M (30 μg) as adjuvant

Serum collected from immunized rabbits (FIG. 54A) or guinea pigs (FIG.54B) was assayed for binding to BG505.SOSIP timer and a V3 peptide. Theimmunization groups elicited comparable anti-BG505 trimer or anti-V3peptide antibodies in rabbits (p<0.05, week 18, Mann Whitney t-test).

Serum collected at weeks 6 and 18 was also tested for neutralizationactivity against a panel of HIV-1 strains (FIG. 55 ). Sera fromimmunized animals were assessed for virus neutralization using a singleround infection assay of TZM-bl cells to determine IC₅₀ values. As shownin FIG. 55 , the BG505 SOSIP DS immunogen elicited an immune responsethat neutralized both autologous virus (BG505.W6M.C2.T332N) and V3directed tier 1 virus (MW965.26) infection, as measured by IC value. Asexpected because rabbits and guinea pigs do not express CD4, theneutralization activity of sera from BG505 SOSIP DS and BG505 SOSIPimmunized animals was similar.

Methods

Immunogen preparation. HIV-1 Env timer preparation was performedsubstantially as described in Example 2. The BG505.SOSIP.664 HIV-1 Envectodomain trimer (SEQ ID NO: 3) or the BG505.SOSIP.664.201C-433C HIV-1Env ectodomain trimer (SEQ ID NO: 26) were used. Trimers were purifiedby affinity chromatography over a VRC01 column, purified by gelfiltration over a Superdex 200 16/60 (GE Healthcare) column in buffercontaining 5 mM HEPES 7.5, 150 mM NaCl, and 0.02% NaN3, and finally,passed through a 447-52D column to remove aberrant trimer species (asdescribed in Example 2 and illustrated in FIG. 36 ). The materials usedfor immunization were tested using Meso-ScaleDiscovery-Electrochemiluminescence Immunoassay (MSD-ECLIA) Analysis asdescribed in Example 2 to confirm antigenic properties (binding tobroadly neutralizing antibodies).

Immunizations. Guinea pig injections consisted of 25 μg of Env trimerformulated in a 400 ul volume in PBS, with 100 μg of PolyIC adjuvant(HMW, Invivogen) or 25 μg Matrix M (Novavax; see Reimer et al., PLoSOne, 7(7):e41451, 2012). For rabbit studies, 30 μg of Env trimer wasformulated with 30 μg Matrix M in a 1 ml volume in PBS. The immunizationwas administered intramuscularly as two separate injections into eachquadriceps. PolyIC adjuvant was prepared by making a 2 mg/ml stocksolution in saline, heating the necessary amount for 10 min at 70° C.,and cooling at room temperature for 1 hour prior to injection.Immunizations were performed on weeks 0, 4, and 16. Blood draws forimmune assessment included a prebleed −1 week sample, followed by blooddraws 2 weeks after each immunization. Collected sera were heatinactivated for 1 hour at 56° C. before being analyzed.

Enzyme-Linked Immunosorbent Assays (ELISAs) for Anti-V3 PeptideResponses. 96 well plates (Reacti-Bind®, Pierce) were coated with 100μl/well of 2 μg/ml peptide in PBS overnight at 4° C. (MW965.26 V3peptide: TRPNNNTRKSIRIGPGQTFYATG (residues 265-287 of SEQ ID NO: 2);BG505 V3 peptide: TRPNNNTRKSIRIGPGQAFYATG (residues 296-318 of SEQ IDNO: 2), amino acid difference underlined). For each consecutive stepfollowing coating, plates were washed 5 times with PBS-T (PBS+0.05%tween) and incubated at 37° C. for 1 hour. After coating, plates wereblocked with 200 μl/well of blocking buffer (B3T: 150 mM NaCl, 50 mMTris-HCl, 1 mM EDTA, 3.3% fetal bovine serum, 2% bovine albumin, 0.07%Tween 20, 0.02% thimerosal). Next, guinea pig sera was diluted in B3Tand added in 5-fold serial dilutions to the plates. Then goatanti-guinea pig antibody conjugated with horseradish peroxidase (KPL,Gaithersburg, Md.,) at a 1:10,000 dilution in B3T was added to eachwell. TMB substrate (SureBlue™, KPL, Gaithersburg, Md., cat #52-00-03)was used to develop plates for 10 minutes before 1N sulfuric acid wasadded to stop the reaction without washing beforehand. Plates were readat 450 nm (Molecular Devices, SpectraMax using SoftMax Pro 5 software)and the final optical density was determined after the horseradishperoxidase nonspecific background binding was subtracted.

D7324-Capture Enzyme Linked Immunosorbent Assays (ELISAs) for Anti-BG505Envelope Trimer Responses. 96 well plates (Reacti-Bind®, Pierce) werecoated with 100 μl/well of 2 μg/ml of anti-D7324 antibody (AaltoBioreagents, Dublin, Ireland) in PBS overnight at 4 degrees Celsius.After coating, plates were blocked for 1 hour at room temperature withPBS+5% skim milk (Difco, Bectin, Dickinson and Company). Plates werethen washed five times with PBS-T (PBS+0.2% tween-20) before adding 0.5μg/ml of BG505 SOSIP.664-D7324 trimer diluted in PBS+10% FBS for twohours at room temperature. After the addition of the trimer, subsequentprocedures mimic the anti-V3 ELISAs except dilutions were made in PBS-Tinstead of B3T.

HIV-1 Neutralization Assays. Sera from immunized animals were assessedfor virus neutralization using previously described methods (Li et al.,J. Virol., 79, 10108-10125, 2005). In the single round infection assay,a reduction in a luciferase luminescence indicates neutralizationactivity. Target cells were TZM-bl cells, which are a clonal HeLa cellline expressing CD4, CXCR4 and CCR5. Upon infection, the HIV-1 viralprotein Tat induces a luciferase reporter gene, whose expression ismeasured as relative light units. Data are represented as the inhibitoryreciprocal dilutions of sera required to inhibit either 50% of infection(IC₅₀), calculated using a regression fit as previously described.

Example 12 Membrane Anchored HIV-1 Env Ectodomain Trimers

This example illustrates production and antigenicity of exemplary HIV-1Env ectodomain trimers stabilized in a prefusion mature closedconformation that include a transmembrane domain for anchoring to thecell surface. Numerous HIV-1 Env ectodomain trimers stabilized in aprefusion mature closed conformation were linked to a transmembranedomain and expressed in cells for anchoring to the cell membrane, aslisted below. Variation within the membrane anchored sequences includesstrain (including chimeras), single chain or not (e.g., sc15ln (15 A.A.linker between gp120/gp41), sc101n (10 A.A. linker between gp120/gp41)),stabilizing mutations (e.g., SOS, DS, IP, or combinations thereof),linker between position 664 and the TM domain (e.g., MPER sequence, or10 ln (10 A.A. linker)), TM domain (e.g, HA or HIV-1 TM), and presenceor absence of a cytoplasmic tail. Exemplary constructs are listed below

TH966.8-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1765)

6545.V4.C1-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1766)

R2184.c4-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1767)

ZM197.7-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1768)

ZM106.9-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1769)

ZM53.12-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1770)

R2184.c4-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1771)

CNE55-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1772)

6545.V4.C1-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1773)

DU422.01-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1774)

25925-2.22-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1775)

CNE58-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1776)

16055-2.3-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1777)

TH966.8-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1778)

ZM55.28a-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1779)

ZM53.12-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1780)

B1369.9A-chim_sc10ln-IP-10ln-HATM (SEQ ID NO: 1781)

ZM197.7-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1782)

16055-2.3-chim_sc10ln-IP-MPER-TM (SEQ ID NO: 1783)

ZM55.28a-chim_sc15ln-SOS-DS-10ln-HATM (SEQ ID NO: 1784)

A FACS-based assay was used to interrogate the antigenicity of themembrane anchored HIV-1 Env ectodomain trimers. Briefly, expressionvectors encoding an immunogen of interest were transfected into cells ina 96-well format. The cells were harvested 2 or 3 days followingtransfection, and stained with antibodies specific for trimeric HIV-1Env (such as VRC26, PGT145, or PGT151), non-trimer specific but broadlyneutralizing antibodies (such as VRC01 or PGT128) and non-trimerspecific poorly neutralizing antibodies (such as 447-52D). The cellswere then stained with appropriate secondary antibody, and analyzed byFACS to assay for antigenicity and expression.

FIG. 56 lists the antigenic characteristics of the constructs listedabove. The designed with the most desirable antigenic characteristicswere those with a single chain HIV-1 Env ectodomain including a 10 aminoacid peptide linker between gp120 and gp41, the “IP” substitution, and a10 amino acid peptide linker or the native MPER sequence between residue664 of gp41 and a TM domain, such as the HA TM domain or the HIV-1 EnvTM domain.

Example 13 HIV-1 Env Ectodomain Trimer Immunogens Based on Ontogeny ofBroadly Neutralizing Antibodies

This example illustrates chimeric HIV-1 Env ectodomain trimer immunogensstabilized in the prefusion mature closed conformation that include aV1V2 domain sequence that (1) bind to mature broadly neutralizingantibodies that target the V1V2 domain, as well as immature somaticprecursors thereof, and that (2) are from a strain of HIV-1 that can beneutralized by V1V2-directed broadly neutralizing antibodies produced bymultiple donors.

Antibodies capable of neutralizing a majority of circulating HIV-1isolates develop in approximately half of those infected with HIV-1 forover five years (Hraber, P. et al. AIDS 28, 163-9 (2014). Intenseinterest has focused on these antibodies, as they provide clues to howan effective vaccine might be developed (Burton, D. R. et al. NatImmunol 5, 233-6 (2004); Haynes, B. F., Kelsoe, G., Harrison, S. C. &Kepler, T. B. Nature biotechnology 30, 423-33 (2012). In specific,broadly neutralizing antibodies (bNAbs)—that arise in multiple donorsand share common features of Env recognition and B-cell ontogeny—mayhave utility as vaccine templates, due to the potential for similarantibodies to be elicited by a common immunogen (or common set ofimmunogens) in the general population (Kwong, P. D. & Mascola, J. R.Immunity 37, 412-25 (2012), Jardine, J. et al. Science 340, 711-6(2013).

An increasing number of such “multidonor” bNAbs have been identified,such as those of the VRC01 class (named for the first antibody of theclass), which share ‘class’ features of molecular recognition and B-cellontogeny (Scheid, J. F. et al., Science 333, 1633-7 (2011); Wu, X. etal., Science 333, 1593-602 (2011); Zhou, T. et al. Immunity 39, 245-58(2013); Zhou, T. et al., Cell 161, 1280-92 (2015)). This commonality hasmotivated the development of immunogens, designed to targetclass-specific features of recognition and to overcome class-specificroadblocks in developmental ontogeny, and success with this strategy hasbeen achieved with immunogens capable of priming the initial stage ofVRC01-class development in mouse models (Dosenovic, P. et al. Cell 161,1505-15 (2015); Jardine, J. G. et al. Science 349, 156-61 (2015), eachof which is incorporated by reference herein).

Structures of the ligand-free forms of these antibodies reveal aprotruding third heavy chain complementarity determining region (CDRH3), which is anionic, often tyrosine sulfated, and critical for Envinteraction. The epitope appears to be quaternary in nature and toinclude an N-linked glycan at residue 160 along with strand C of V1V2.In terms of B-cell ontogeny, approximations of the unmutated commonancestor (UCA) have been inferred for V1V2-directed bNAb lineages fromdonors CH0219 and CAP256 (Bonsignori, M. et al. J Virol 85, 9998-10009(2011); Doria-Rose, N. A. et al. Nature 509, 55-62 (2014), each of whichhis incorporated by reference herein), which indicate the long anionicCDR H3 to be a product of recombination. Initial recognition of UCA (orof V-gene reverted approximations) appears to be restricted to selectstrains of HIV-1 (e.g. CAP256-SU or ZM233), to use similar D genes andin some cases related V genes, and to contain similar motifs (e.g. YYD)in the CDR H3.

While antibodies against the same supersite of HIV-1 vulnerability oftenshow diverse modes of recognition, bNAbs against the membrane-distalV1V2 apex of pre-fusion closed conformation of HIV-1 Env appear to sharea number of characteristics. Thus far, V1V2-directed bNAbs have beenidentified in four donors: the CH0219 donor, with bNAbs CH01-CH04(Bonsignori, M. et al. J Virol 85, 9998-10009 (2011)); the CAP256 donor,with bNAbs CAP256-VRC26.01-12 (Doria-Rose, N. A. et al. Nature 509,55-62 (2014); the IAVI 24 donor, with bNAbs PG9 and PG16 (Walker, L. M.et al. Science 326, 285-9 (2009); and the IAVI 84 donor with bNAbsPGT141-145 (Walker, L. M. et al. Nature 477, 466-70 (2011) andPGDM1400-1412 (Sok, D. et al. Recombinant HIV envelope trimer selectsfor quaternary-dependent antibodies targeting the trimer apex. Proc NatlAcad Sci USA 111, 17624-9 (2014).

Neutralization screening with UCA and intermediates of V1V2-directedbNAbs was used to engineer antigens capable of interacting withdevelopmental intermediates. Altogether the structural similarities inantibody recognition along with ontogeny similarities (and differences)in development indicate the V1V2-directed bNAbs to form an ‘extendedclass’, which do not necessarily share genetic commonalities, butnonetheless display a characteristic mode of antigen interaction.Extended-class immunogens—such as the soluble chimeric trimers disclosedherein through an ontogeny-based chimera strategy-provide a generalmeans for eliciting bNAbs against specific sites of Env vulnerability.

V1V2 Chineras

Several HIV-1 Env molecules that specifically bind to mature broadlyneutralizing antibodies that target the V1V2 domain, as well as immaturesomatic precursors thereof, were identified (FIG. 57 ). These includeEnv proteins from the CAP256.SU, BB201.B42, KER2018.11, CH070.1,ZM233.6, Q23.17, A244, T250-4, and WITO.33 strains of HIV-1. To identifythe HIV-1 strains shown in FIG. 57 , mature and reverted V1V2-directedbNAbs were tested for neutralization across ˜200 exemplary HIV-1isolates. The V1V2 sequence of each of the HIV-1 strains (positions126-196, HXB2 numbering) is shown, with the A, B, C, C′ and D secondarystructures indicated.

With regard to FIG. 57 , inferred ancestor and intermediates ofV1V2-directed bNAbs neutralize a common set of HIV-1 isolates. Matureand reverted V1V2-directed bNAbs were tested for neutralization across˜200 HIV-1 isolates. “Neutralized” represents the number of HIV-1strains with IC₅₀ of less than 50 mg/ml, and “Total” indicates thenumber of HIV-1 strains tested; IC₅₀ for select strains is indicated byan enlarged colored dot. The reverted antibodies were previouslyidentified, see, e.g., Bonsignori et al., J. Virol., 85(19):9998-10009,2011, which is incorporated by reference herein.

HIV-1 strains neutralized by the broadly neutralizing antibodyrevertants shown in FIG. 57 were ranked according to probabilitiesobtained by frequentist analysis, for which enhancement in likelihood ofinteraction with the earliest neutralizers is provided. The foldenhancement of interaction probability for the selected strains relativeto a random strain from the ˜200-virus panel using a frequentistapproach is shown in the following table, as evaluated based on (i) all13 antibodies in FIG. 57 , (ii) mature antibodies (CH04, PG9,CAP256-CAP256.08, and PGT145), (iii) non-mature antibodies, (iv)germline or UCA antibodies (CH0219-UCA, PG9-gHgL, PGT145-gHgL,CAP256-VRC26.UCA), (v) intermediate antibodies (non-mature ornon-germline/UCA), and (vi) earliest neutralizers (CH0219-UCA, PG9-gHgL,PGT145-gHL, CAP256-VRC26.I1). The enhancement of interaction probabilityfor using nine selected strains compared to a random strain is alsoshown.

Mature Non-Mature Intermediate Germline and Earliest Strain All Abs AbsAbs Abs UCA Abs neutralizers Fold enhancement of interaction probabilityfor each selected strain WITO.33 2.07 1.23 4.28 3.45 14.98 23.02 ZM233.62.24 1.63 4.28 2.3 29.96 23.02 T250-4 2.36 1.63 4.28 4.6 0 11.51 CH070.11.77 1.23 3.21 2.3 14.98 11.51 BB201.B42 2.07 1.63 3.21 2.3 14.98 11.51KER2018.11 2.07 1.63 3.21 2.3 14.98 11.51 Q23.17 2.07 1.63 3.21 2.314.98 11.51 A244 2.36 1.63 4.28 3.45 14.98 11.51 CAP256.SU 2.07 1.633.21 3.45 0 11.51 Fold enhancement of interaction probability for usingthe nine selected strains 3.25 1.63 7.48 5.76 29.96 46.04

Chimeric HIV-1 Env ectodomain timer immunogens were produced thatinclude the V1V2 domain sequence (positions 126-196) of the CAP256.SU,BB201.B42, KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, orWITO.33 strains of HIV-1, with the remainder includingBG505.SOSIP.DS.368R sequence (including gp120 positions 31-125 and197-511 and gp41 positions 512-644), as follows: Q23.17 chimera (SEQ IDNO: 2126), ZM233.6 chimera (SEQ ID NO: 2125), WITO.33 chimera (SEQ IDNO: 2128), A244 chimera (SEQ ID NO: 2127), BB201.B42 chimera (SEQ ID NO:2122), KER2018.11 chimera (SEQ ID NO: 2123), CH070.1 chimera (SEQ ID NO:2124), CAP256.SU chimera (SEQ ID NO: 2121), and T-250-4 chimera (SEQ IDNO: 2129).

The transplanted V1V2 region is illustrated in FIG. 58A. FIG. 58B showsan exemplary gel filtration and negative stain EM (2D class averages) ofa representative chimera, BG505 SOSIP.664.DS.368R.CAP256-SU. The bindingof the soluble chimeric HIV-1 Env ectodomains to the ancestors,intermediates, and mature V1V2-directed bNAbs listed in FIG. 57 wasassayed by ELISA (FIG. 58C). Additionally, these chimeric HIV-1 Envectodomain trimers were incubated with a molar excess of soluble CD4 andtested for binding to 17b mAb—none of the chimeric HIV-1 Env ectodomaintrimers specifically bound to 17b in the presence of sCD4. Further, eachof the chimeric HIV-1 Env ectodomain trimers did not specifically bindto the poorly neutralizing antibody 447-52D, but did specifically bindto V1V2 targeted broadly neutralizing antibodies such as PGT145, VRC26,PGT121, and/or 2G12.

426c and d45-01dG5 Chimeras

Additional chimeras were generated that combine the BG505 “platform”described in Example 5, with a V1V2 domain from a CAP256.SU, BB201.B42,KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, or WITO.33 strain asdescribed in this example, with the remainder of the gp120 portion ofthe HIV-1 Env ectodomain from a HIV-1 Env molecule known to interactwith mature and UCA forms of VRC01-class antibodies (such as Env fromthe clade C 426c strain with N276D, N460D, N463D substitutions (see Wuet al., Cell, 161, 470-485, 2015, which is incorporated by referenceherein), or Env from the clade B d45-01dG5 strain (see McGuire et al., JExp. Med., 210, 655-663, 2013, incorporated by reference herein).

The amino acid sequence of 426c Env with N276D, N460D, N463Dsubstitutions is provided as SEQ ID NO: 2144. The amino acid sequence ofd45-01dG5 Env is provided as SEQ ID NO: 2145. These ectodomain trimershave unique antigenic characteristics that provide for binding to matureand UCA forms of multiple classes or broadly neutralizing antibodies(targeting the CD4 binding site and the V1V2 domain) and can be used toinduce an immune response to HIV-1 Env in a subject. These immunogensare of particular interest for use as a “prime” immunogen in aprime-boost immunization protocol for eliciting an immune response toHIV-1 Env.

The chimeric Env ectodomains included the sequences from the following(HXB2 numbering):

For the 426c chimera:

BG505 “platform”: 31-45 and 478-507, 512-664;

V1V2 domain: positions 126-196 from CAP256.SU, BB201.B42, KER2018.11,CH070.1, ZM233.6, Q23.17, A244, T250-4, or WITO.33

426c: 46-125, 197-478

This chimeric Env ectodomain further included SOSIP substitutions, DSsubstitutions (201C/433C), and N276D, N460D, N463D substitutions toeliminate the glycosylation sites at positions 276, 460, 463.

For the d45-01dG5 chimera:

BG505: 31-45 and 478-507, 512-664 with SOSIP substitutions;

V1V2 domain: 126-196 positions 126-196 from CAP256.SU, BB201.B42,KER2018.11, CH070.1, ZM233.6, Q23.17, A244, T250-4, or WITO.33

This chimeric Env ectodomain further included SOSIP substitutions and DSsubstitutions (201C/433C). The donor 45_01dG5 Env naturally lacks aglycan at 276 and 460 and this is sufficient to allow UCA forms ofVRC-01 class antibodies to bind. Specific examples of sequences of suchchimeric HIV-1 Env ectodomains are provided as SEQ ID NOs: 2146-2159.

As proof-of-principle, a chimera was generated that includes the BG505“platform” (positions 31-45 and 478-507, 512-664), with the remainder ofgp120 from the 426c strain with mutations of the glycan sequon atpositions 276, 460 and 463, and the “DS” substitutions (201C/433C) andtested antigenically (FIG. 60 ). This construct bound to VRC20 gHgL andVRC01 gHgL UCA antibodies, as well as the indicated neutralizingantibodies.

UCA and intermediate matured antibodies related to known broadlyneutralizing antibodies that can be used to interrogate the antigenicityof the disclosed chimeras are known. Non-limiting examples include UCAand intermediate matured antibodies related to VRC26, PGT145, CH01, PG9(see, e.g., Alam et al., PNAS, 110, 18214-9, 2013; Bonsignori et al, JVirol, 85, 9998-10009, 2011; Bonsignori et al, J Virol, 86, 4688-4692,2012; doria-rose et al, Nature, 509, 55-62, 2014; Pancera et al., JVirol, 84, 8098-8110, 2010; Walker et al., Nature, 477, 466-470, 2011;each of which is incorporated by reference herein) and VRC01 (see, e.g.,Jardine, J. et al. Science 340, 711-6, 2013, which is incorporated byreference herein).

Example 14 Recombinant HIV-1 Env Ectodomain Trimers Including BG505 andJRFL Sequences

This example describes chimeric HIV-1 Env ectodomain trimers thatinclude chimeric sequences having a BG505 “platform,” as well as otherBG505 structural elements (such as a V1, V2, and/or V3 domain), with theremaining sequence of the HIV-1 ectodomain based on the JRFL strain ofHIV-1.

Chimeric HIV-1 Env ectodomains were expressed that include HIV-1 Envpositions 31-507 joined by a 6R cleavable linker to gp41 positions512-664. The BG505 sequence was used for gp120 positions 31-45 and478-507 and gp41 residues 512-664. Some constructs also included BG505residues for “Interface Residue Set A” or “Int. Res. Set A:” gp120positions 46-54, 70-75, 84-89, 99, 102, 106, 107, 114, 215, 220-224,226, 244, 471-473, and 476-477. Some constructs also included BG505sequence for particular structural elements, such as the:

V1 loop (gp120 positions 119-153)

V2 loop (gp120 positions 154-205)

Strand C of the V1V2 domain (gp120 positions 166-173)

V3 domain (gp120 positions 296-331)

Positions 191-205

V2 loop and V3 domain

Strand C and V3 domain

Positions 191-205 and Strand C

V1 and V3

V1, Strand C, and V3

V1, V2, and V3

HIV1 Env positions 191-205 are a set of residues in strand D of the V1V2which forms extensive contacts with the B20-B21 sheets and may help tostabilize the chimeric JRFL molecule. The recombinant HIV-1 Env proteinsalso included the SOSIP and 201C/433C substitutions (for stabilization)and an E168K substitution to maximize binding to V1V2-directed broadlyneutralizing antibodies.

The JRFL sequence was used for the remaining HIV-1 Env sequence. Thefollowing recombinant HIV-1 Env ectodomain trimers were expressedaccording to methods described in Examples 1 and 2, and theirantigenicity was assayed using a panel of antibodies (FIG. 59 ).

SEQ Name Chimeric positions ID NO JRFLgp140.6R.SOSIP.664.E168K, BG505gp41 BG505: gp120 positions 31-45 and 478-507, V2, V3, gp41 1732 chim,I201C, A433C,_v2_v3 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, V3; gp41 512- 1735 chim, I201C, A433C,_v3 664; JRFL:remainder JRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120positions 31-45 and 478-507, Int. Res. Set A, 1736 chim, +int I201C,A433C,_strC_v3 Strand C, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, Int. Res. Set A, 1738 chim, +int I201C, A433C,_191-205_v3positions 191-205, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, Int. Res. Set A, 1739 chim, +int I201C, A433C,_v1_v3positions 191-205, V1, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, Int. Res. Set A, 1741 chim, +int I201C, A433C,_v3 V3; gp41512-664; JRFL: remainder JRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505:gp120 positions 31-45 and 478-507, Int. Res. Set A, 1742 chim, +intI201C, A433C,_v2_v3 V2, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, Int. Res. Set A, 1744 chim, +int I201C, A433C,_v1_strC_v3V1, Strand C, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, V1, V3; gp41 1758 chim, I201C, A433C,_v1_v3 512-664; JRFL:remainder JRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120positions 31-45 and 478-507, Int. Res. Set A, 1759 chim, +int I201C,A433C,_v1_191-205_v3 V1, positions 191-205, V3; gp41 512-664; JRFL:remainder JRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120positions 31-45 and 478-507, positions 191- 1760 chim, I201C,A433C,_191-205_v3 205, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, Int. Res. Set A, 1761 chim, +int I201C,A433C,_strC_191-205_v3 Strand C, positions 191-205, V3; gp41 512-664;JRFL: remainder JRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120positions 31-45 and 478-507, Int. Res. Set A, 1762 chim, +int I201C,A433C,_v1_v2_v3 V1, V2, V3; gp41 512-664; JRFL: remainderJRFLgp140.6R.SOSIP.664.E168K, BG505 gp41 BG505: gp120 positions 31-45and 478-507, V1, Strand C, V3; 1763 chim, I201C, A433C,_v1_strC_v3 gp41512-664; JRFL: remainderEach of the recombinant HIV-1 Env ectodomain trimers listed in the abovetable also included the SOSIP, R6, 664, E168K, 1201C, and A433Csubstitutions.

As illustrated in FIG. 59 , each of these recombinant HIV-1 Envectodomain trimers has substantially reduced binding to 17b antibody inthe presence of sCD4 compared to control BG505.SOSIP, and bound toprefusion mature closed conformation targeted antibodies, such asPGT145. The binding assays were performed as described in Examples 1 and2. The reduced binding to VRC26 of JRFL based constructs is due to thefact that this antibody interacts poorly with JRFL Env.

Example 15

The following table (Table 13) provides a description of sequencesprovided herein. Sequence of particular interest for use as immunogensto induce an immune response to HIV-1 Env are marked with a

5 Mutations 1 4 Compared SEQ Native to 6 ID 2 3 Back- Native Additional7 NO Code Name ground Background mutations Comment 0001 0 HXB2VIRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTD LKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEE WIRSVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSN NTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQA RQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNIT NWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSA VSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL 0002 0 BG505 BG505MRVMGIQRNCQHLFRWGTMILGMIIICSAAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVT NNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVM IRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCTVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTG SNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLL SGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLW YIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELK ISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL 0003 A001 BG505 SOSIPBG505 SOSIP 0004 A002 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y191FStabilized V1V2 cap T332N_ T332N Y191F 0005 A003 BG505, SOSIP, R6, 664,BG505 SOSIP, R6, 664, Y191W Stabilized V1V2 cap T332N_ T332N Y191W 0006A004 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A433P Disrupt CD4-boundT332N_ T332N sheet conf. A433P 0007 A005 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Q432P Disrupt CD4- T332N_ T332N bound sheet conf. Q432P0008 A006 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S174C/A319CDS (disulfide) T332N_ T332N S174C/A319C 0009 A007 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, L175C/T320C DS T332N_ T332N L175C/T320C 0010 A008BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P220C/A578C DS T332N_ T332NP220C/A578C 0011 A009 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,A221C/A582C DS T332N_ T332N A221C/A582C 0012 A010 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, A200C/P313C DS T332N_ T332N A200C/P313C 0013 A011BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 498C/W610C DS T332N_ T332N498C/W610C 0014 A012 BG505.IP.R6.664. BG505 IP, R6, 664, G41C/Q540CDS-non SOS context T332N_ T332N G41C/Q540C 0015 A013 BG505.IP.R6.664.BG505 IP, R6, 664, P43C/A526C DS-non SOS context T332N_ T332N P43C/A526C0016 A014 BG505.IP.R6.664. BG505 IP, R6, 664, A221C/A582C DS-non SOST332N_ T332N A221C/A582C 0017 A015 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, G527C/N88C DS T332N_ T332N G527C/N88C 0018 A016BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q540C/P43C DS T332N_ T332NQ540C/P43C 0019 A017 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,E164C/N197C DS T332N_ T332N E164C/N197C 0020 A018 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, P124C/R166C DS T332N_ T332N P124C/R166C 0021 A019BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, D18OC/I423C DS T332N_ T332ND18OC/I423C 0022 A020 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,N195C/I423C DS T332N_ T332N N195C/I423C 0023 A021 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, N195C/A433C DS T332N_ T332N N195C/A433C 0024 A022BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S199C/A433C DS T332N_ T332NS199C/A433C 0025 A023 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,5199C/G431C DS T332N_ T332N S199C/G431C 0026 A024 *BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, I201C/A433C DS T332N_ T332N I201C/A433CAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKV YSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRP NNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQcMYA PPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV0ARNLLSGIVQQQSNLLRAPEAQQHLLKLT VWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 0027 A025 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, T320C/P438C DS T332N_ T332N T320C/P438C 0028 A026BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, D180C/K421C DS T332N_ T332ND180C/K421C 0029 A027 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M530WCavF at gp41- T332N_ T332N tryptophan clasp. M530W Stabilize gp41-tryptophan clasp interactions 0030 A028 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, F159Y CavF at gp120 V1/V2. T332N_ T332N Substitute Y orF159Y W, stabilize V1/V2/V3 0031 A029 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, F223W CavF at gp120 beta-5. T332N_ T332N Substitute Y orF223W W. stabilize gp120/gp41 interface 0032 A030 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, L544Y, F223W CavF at gp41 tip of T332N_ T332Nα-6. 544: F, Y, or L544Y_F223W W 223: Y or W. stabilize gp120/gp41interface 0033 A031 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L523FFusion Peptide T332N_ T332N Cavity Fill L523F 0034 A032BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F522Y Fusion Peptide T332N_T332N Cavity Fill F522Y 0035 A033 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, W35Q Exposed Nterm T332N_ T332N hydrophobic W35Qresurfaced 0036 A034 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,A200C/P313C, nonSOS_multiple T332N_ T332N A221C/A582C cyscysA200C/P313C_A221C/ A582C 0037 A035 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Q432E Disrupt CD4-bound T332N sheet conf. T332N_ Q432E0038 A036 BG505.SOSIP. BG505 SOSIP, R6, 664, Q432D Disrupt CD4-boundR6.664. T332N sheet conf. T332N_ Q432D 0039 A037 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, M434P Disrupt CD4-bound T332N sheet conf. T332N_M434P 0040 A038 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y435PDisrupt CD4-bound sheet conf. T332N_ T332N Y435P 0041 A039BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P436PDisrupt CD4-bound sheet conf. T332N_ T332N A436P 0042 A040BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P437A proline removal T332N_T332N P437A 0043 A041 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P438Aproline removal T332N_ T332N P438A 0044 A042 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, P437A, P438A proline removal T332N_ T332N P438A.P437A0045 A043 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F139W, I326RCavF at the interface of V1V2 and V3 T332N_ T332N loops. Sustitute-T139W.I326R T139: F, M, I, Y; I326: M, W, F, Y. stabilize V1V2/V3loop interactions in mature closed state 0046 A044 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, T179W CavF at V1V2 loop gp120 core T332N_ T332Ninterface. L179W Substitute-F, Y M, I.stabilize interaction between V1V2 loop and gp120 core in mature closedstate 0047 A045 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y39F, S534VCavF. add hydrophobicity at T332_NY39F.S534V T332Ngp120/gp41 interface Substitute- S534: I, W, F, A, M; Y39: W, M, I 0048A046 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y39W, S534ACavF. add hydrophobicity at T332N_ T332N gp120/gp41 interface. AY39W.S534A 0049 A047 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,Y39F, S534V, T37V, T499V CavF. add hydrophobicity at T332N_ T332Ngp120/gp41 interface 39F.S534V.T37V.T499V 0050 A048 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, Y39F, Y40F, S534V, CavF. add hydrophobicity atT332N_ T332N T37V, T499V gp120/gp41 interface Y39F.Y40F.S534V.T37V.F499V 0051 0 CAP256.SU CAP256.SU 0052 A049 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, N425C/I430C S-Stabilizes CD4 binding loop T332N_ T332NN425C I430C 0053 A050 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,Y318C/437C, G473A DS-Restrain PGT122 bound T332N_ T332N conformation;Y318C P437C G473A reduce CD4 binding 0054 A051 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, M426W CavF at CD4bs. Substitute-F, Y, T332N_ T332NL, V, I. M426W Constrains CD4 binding loop 0055 A052 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, S174C/A319C, G473A DS-Restrain PGT122 bound T332N_T332N conformation; S174C A319C G473A reduce CD4 binding 0056 A053BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L175C/T320C, G473ADS-Restrain PGT122 bound T332N_ T332N conformation; L175C T320C G473Areduce CD4 binding 0057 A054 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,F176C/D180C DS-Restrain PGT122 bound T332N_ T332N conformationF176C D180C 0058 A055 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,A204C/A436C, G473A DS-Restrain PGT122 bound T332N_ T332N conformation;A204C A436C G473A reduce CD4 binding 0059 A056 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A204C/M434C, G473A DS-Restrain PGT122 bound T332N_ T332Nconformation; A204C M434C G473A reduce CD4 binding 0060 A057BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P212C/K252CDS-Restrain PGT122 bound T332N_ T332N conformation P212C K252C 0061 A058BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P220C/A200CDS-Restrain PGT122 bound T332NP220CA200C T332N conformation 0062 A059BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G314C/A200CDS-Restrain PGT122 bound T332N_ T332N conformation G314C A200C 0063 A060BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A204C/M434CDS-Restrain PGT122 bound T332N_ T332N conformation A204C M434C 0064 A061BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L122C/L125CDS-Restrain PGT122 bound T332N_ T332N conformation L122C L125C 0065 A062BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G473ACavF at CD4bs. Substitute-any. T332NG473A T332N sterically interferewith CD4 binding, without affecting Ab binding 0066 A063BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G473S Same as Seq_0065 T332N_T332N G473S 0067 A064 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G473YSame as Seq_0065 T332N_ T332N G473Y 0068 A065 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, G431P Same as Seq_0065 T332N_ T332N G431P 0069 A066BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, N425C/A433CDS-Fixes PGT122 bound state T332N_ T332N N425C A433C 0070 A067BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120C/Q315CDS-Fixes PGT122 bound state T332N_ T332N V120C Q315C 0071 A068BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P124C/T164CDS-Fixes PGT122 bound state T332N_ T332N P124C T164C 0072 A069BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T128C/T167C, G473ADS-Restrain PGT122 bound T332N_ T332N conformation; T128C T167C G473Areduce CD4 binding 0073 A070 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,I424C/F382C DS-Fixes PGT122 bound state T332N_ T332N I424C F382C 0074A071 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R298C/A329CDS-Fixes PGT122 bound state T332N_ T332N R298C A329C 0075 A072BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M426PCavF at CD4bs. Substitute-P. T332N_ T332N Rigidities CD4 binding loopM426P 0076 A073 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y191W G473ACavF at CD4bs and V1/V2 cap T332N_ T332N Y191W G473A 0077 A074BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q203C/1122CDS-Fixes PGT122 bound state T332N_ T332N Q203C L122C 0078 A075BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M426ACavF at CD4bs. Substitute-G, V, T332N_ T332NI, L, I, Y, W, R, K. prevents M426A triggering on CD4 binding 0079 A076BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y191W A433PCavF at CD4bs and V1/V2 cap T332N_ T332N Y191W A433P 0080 A077BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T372C/S364CDS-Fixes PGT122 bound state T332N_ T332N T372C S364C 0081 0 BB201.B42BB201.B42 0082 A078 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V36C/V608CDS-Fixes PGT122 bound state T332N_ T332N V36C V608C 0083 A079BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M426FCavF at CD4bs. Substitute-Y, W, T332N_ T332N L, I, V, R, K, G, A. BlocksM426F transition to CD4 bound conformation 0084 A080 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, W69P helix 0 disruption T332N_ T332N W69P 0085A081 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V68P helix 0 disruptionT332N_ T332N V68P 0086 A082 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,T71P helix 0 disruption T332N_ T332N T71P 0087 A083 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, H66C/K207C disulfide T332N_ T332N H66C/K207C 0088A084 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A73C/G572C disulfideT332N_ T332N A73C/G572C 0089 A085 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, F53C/G575C disulfide T332N_ T332N F53C/G575C 0090 A086BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V75WCavF at gp120/gp41 interface T332N_ T332N V75W 0091 A087BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V75FCavF at gp120/gp41 interface T332N_ T332N V75F 0092 A088BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V75MCavF at gp120/gp41 interface T332N_ T332N V75M 0093 A089BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V208WCavF between α1 and the strand T332N_ T332N leading into what V208Wforms the bridging sheet in the CD4-bound form.Destabilize CD4-bound state 0094 A090 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, V208Y Same as Seq_0093 T332N_ T332N V208Y 0095 A091BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V208F Same as Seq_0093 T332N_T332N V208F 0096 A092 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V208MSame as Seq_0093 T332N_ T332N V208M 0097 A093 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A58C/T77C helix 0 disruption T332N_ T332N A58C/T77C 0098A094 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, D57C/T77Chelix 0 disruption T332N_ T332N D57C/T77C 0099 A095 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V68C/S209C helix 0 disruption T332N_ T332NV68C/S209C 0100 A096 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V68C/208Chelix 0 disruption T332N_ T332N V68C/V208C 0101 A097 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V66C/S209C helix 0 disruption T332N_ T332NV66C/S209C 0102 A098 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V67Phelix 0 disruption T332N_ T332N N67P 0103 A099 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, N66P helix 0 disruption T332N_ T332N H66P 0104 A100BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, N67P/H66P helix 0 disruptionT332N_ T332N N67P/H66P 0105 A101 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A58C/T77C, N67P, H66P helix 0 disruption T332N_ T332NA58C/T77C/N67P/H66P 0106 A102 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,D57C/T77C, N67P, H66P helix 0 disruption T332N_ T332N D57C/T77C/N67P/H66P 0107 0 KER2018.11 KER2018.11 0108 A103 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, V68C/V208C, N67P, H66P helix 0 disruption T332N_ T332NV68C/V208C/N67P/H6 6P 0109 A104 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, V68C/S209C, N67P, H66P helix 0 disruption T332N_ T332NV68C/S209C/N67P/ H66P 0110 A105 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, D474A, R476A Destabilization of CD4 binding site T332N_T332N D474A/R476A 0111 A106 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,W112I Destabilization of CD4 binding site T332N_ T332N W112I 0112 A107BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, W112MDestabilization of CD4 binding site T332N_ T332N WH2M 0113 A108BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, W427IDestabilization of CD4 binding site T332N_ T332N W427I 0114 A109BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, W427MDestabilization of CD4 binding site T332N_ T332N W427M 0115 A110BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R429NDestabilization of CD4 binding site T332N_ T332N R429N 0116 A111BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R429LDestabilization of CD4 binding site T332N_ T332N R429L 0117 A112BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R429L, W427MDestabilization of CD4 binding site T332N_ T332N R429L/W427M 0118 A113BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, D474ADestabilization of CD4 binding site T332N_ T332N D474A 0119 A114BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R476A T332N_ T332N R476A 0120A115 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I201C/A433C, F159YDS and CavF at gp120 V1/V2. T332N_ T332N stabilize V1/V2/V3I201C/A433C_F159Y 0121 A116 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,R166CG/V1270 V1V2 disulfide T332N_ T332N stabilization, preventR166CG/V127C v1v2 from adopting cd4-bound- conformation 0122 A117BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G314C/S199C,V1V2V3 disulfide stabilization. T332N_ T332N R166CG/V127Cprevent v1v2 from adopting cd4- G314C/S199C/R166CG/ V127Cbound-conformation 0123 A118 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,K421W, D180L CavF at V1V2 interaction near Res. T332N_ T332N180 with V3 and K421W.D180L base of beta-21, substitute-421: F, W, Y;180: L, V, I, M. prevent v1v2 to V3 along with B21from adopting cd4-bound- conformation 0124 A119 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, G431GC/S199C DS T332N_ T332N G431.GC.S199C 0125A120 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R166C/V127GC DS T332N_T332N R166C.V127GC 0126 A121 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,G314C/S199C DS T332N_ T332N G314C.S199C 0127 A122 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, G314CG/S199C DS T332N_ T332N G314CG.S199C 0128A123 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120WCavF at N-term of β-2. Substitute-F, T332N_ T332NW, Y, L, I, M. β-2 extends in cd4- V120Wbound state, this stabilizes small hydrophobic pocket in ground state0129 A124 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120W, Q203VSame as Seq_0128 T332N_ T332N V120W.Q203V 0130 A125 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, delP124 proline removal. Removal of ground T332N_T332N state destabilization/flexibility deltaP124 0131 A126BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L125W, delP124proline removal and CavF at Cavity T332N_ T332Nbetween N-term of V1V2 domain and L125W_deltaP124V3 near Res. 127 and 126-196 disulfide of V1V2. Substitute-F, W,Y, L, I, M, V. Removal of ground state destabilization/flexibility 0132A127 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R151E, E153W, Q.328WCavF at V1 loop at resi 153. T332N_ T332N Substitute-153:R151E.E153W.Q, 328W F, W, Y, L, I, M, V; 328 F, W, Y, L, I, M, V. Addinghydrophobic patch at V1 loop to V3 loop 0133 A128 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, F159W CavF at Primary hydrophobic pocket T332N_T332N etween V1V2 and V3 near resi 159. F159W Substitute-W or Y.Stabilizing the hydrophobic core of the V1V2-V3 interactions 0134 A129BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F317W Same as Seq_0133 T332N_T332N F317W 0135 A130 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M161WCavF at Hydrophobic patch at Cterm T332N_ T332Nof V1V2 strand B. Substitute- M161W F, W, Y, L, I.Stabilizing strand B to V3 near trimeric interface 0136 A131BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I309W Same as Seq_0135 T332N_T332N I309W 0137 A132 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,L125R, F317D Salt bridge. Salt bridge T332N_ T332N interaction inL125R.F317D hyrophobic shell 0138 A133 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L125WE, F317R Salt bridge. Salt bridge T332N_ T332Ninteraction in L125WE.F317R hyrophobic shell 0139 A134BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S115WCavF at C-term of α-1. Substitute-F, T332N_ T332N W, Y, L, I, M, V. WillS115W fill cavity at top of α-1 which shifts conformation inCD4-bound state 0140 A135 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,P118W CavF/proline removal at C-term of α- T332N_ T332N1. Substitute-F, W, Y, L, P118W I, M, V. Will fillcavity at top of α-1 which shifts conformation in CD4-bound state 0141A136 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 3206Aproline removal. Removal of ground T332N_ T332Nstate destabilization/flexibility P206A 0142 A137 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, delP206 proline removal. Removal of ground T332N_T332N state destabilization/flexibility deltaP206 0143 A138BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A70Y CavF-extension of T332N_T332N hydrophobic/aromatic patch- A70Y gp41/gp120 stabilization(a7/a0cavity close to A70). Substitute-F, Y, W 0144 A139BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A70F Same as Seq_0143 T332N_T332N A70F 0145 A140 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L111YSame as Seq_0143 T332N_ T332N L111Y 0146 A141 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L111F Same as Seq_0143 T332N_ T332N L111F 0147 A142BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T202P disrupt bridging sheet/T332N_ T332N destabilizing T202P CD4 bound state 0148 A143BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120P Same as Seq_0147 T332N_T332N V120P 0149 A144 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120TSame as Seq_0147 T332N_ T332N V120T 0150 A145 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L122K Same as Seq_0147 T332N_ T332N L122K 0151 A146BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P313C/A200C, T51C/K574C DsT332NP313C/A200C/T51C/K5 T332N 74C 0152 A147 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, P313C/A200C, F53C/K574C Ds T332N_ T332NP313C/A200C/F53C/K5 74C 0153 A148 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, 313C/A200C, T51C/A578C Ds T332N_ T332NP313C/A200C/T51C/A5 78C 0154 A149 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, T128C/L165C, P313C/A200C, Ds T332N_ T332N T51C/K574CT128C/L165C/P313C/A 200C/T51C/K574C 0155 A150 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, T128C/L165C, P313C/A200C, Ds T332N_ T332N F53C/K574CT128C/L165C/P313C/A 200C/F53C/K574C 0156 A151 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, T128C/L165C, P313C/A200C, Ds T332N_ T332N F51C/A578CT128C/L165C/P313C/A 200C/T51C/A578C 0157 A152 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, I573T destabilize gp41 helix bundle T332N_ T332N I573T0158 A153 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G594Ndestabilize gp41 helix bundle T332N_ T332N G594N 0159 A154BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I573T-G594Ndestabilize gp41 helix bundle T332N_ T332N I573T-G594N 0160 A155BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I573T-G594N-K574Edestabilize gp41 helix bundle T332N_ T332N I573T-G594N-K574E 0161 A156BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I573T-G594N-K574Tdestabilize gp41 helix bundle T332N_ T332N I573T-G594N-K574T 0162 A157BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A433C/L122C T332N_ T332NA433C L122C 0163 A158 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,Q428C/E560C T332N_ T332N Q428C E560C 0164 A159 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Q428C/A561C T332N_ T332N Q428C A561C 0165 A160BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q428C/Q562C T332N_ T332NQ428C Q562C 0166 A161 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,K574C/D107C T332N_ T332N K574C D107C 0167 A162 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Q575C/Q550C T332N_ T332N Q575C Q550C 0168 A163BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q575C/Q551C T332N_ T332NQ575C Q551C 0169 A164 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,R579C/Q550C T332N_ T332N R579C_Q550C 0170 A165 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, M426C/E370C T332N_ T332N M426C E370C 0171 A166BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M426C/G380C T332N_ T332NM426C_G380C 0172 A167 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T123WCavF at trimer axis. Substitute- T332N_ T332N L, Y, L, M, V. stabilizeT123W prefusion axis interactions 0173 A168 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, I423W, I201W CavF at parallel b-strand #. T332N_ T332NSubstitute- I423W_I201W F, Y, L, M, V. prevent bridging  sheet formation0174 0 CH070.1 CH070.1 0175 A169 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, K117W CavF at trimer axis. T332N_ T332NSubstitute-F, Y, L, K117W I, H, R, E, D, M, V. stabilize prefusionaxis interactions 0176 A170 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,K117E CavF/charged at trimer axis. T332N_ T332NSubstitute-F, Y, L, I, H, K117E R, E, D, M, V. stabilizeprefusion axis interactions 0177 A171 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, K121E CavF/charged at trimer axis. T332N_ T332NSubstitute-F, Y, L, I, H, K121E R, E, D, M, V. stabilizeprefusion axis interactions 0178 A172 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, S110W CavF at trimer axis/gp41 interface T332N_ T332Nboundary. Substitute- S110W F, Y, L, I, M, V. stabilize prefusion axisinteractions/gp41 interface/ prevent helix movement 0179 A173BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q114W Same as Seq_0178 T332N_T332N Q114W 0180 A174 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P220WCavF at gp41 interface. Substitute- T332N_ T332NF, Y, L, I, M, V. stabilize P220W gp41 interface 0181 A175BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T50WCavF at gp41 interface. Substitute- T332N_ T332NF, Y, L, I, M, V. stabilize T50W gp41 interface 0182 A176BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R429WCavF at parallel b-strand #. T332N_ T332N Substitute-F, Y, L, I, M, V.R429W prevent bridging sheet formation 0183 A177 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V120W, I201C/A433Cds/CavF under V1V2 cap. Substitute- T332N_ T332N F, Y, L, I, H, R,V120W_I201C_A433C E, D, M, V. stabilize prefusion axis interactions/combined with DS 0184 A178 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,K121W, I201C/A433C ds/CavF at trimer axis. Substitute- T332N_ T332NF, Y, L, M, V. K121W_I201C_A433C stabilize prefusion axisinteractions/combined with DS 0185 A179 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, T123W, I201C/A433C Same as Seq_0184 T332N_ T332NT123W I201C A433C 0186 A180 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,K117W, I201C/A433C ds/CavF at trimer axis. Substitute- T332N_ T332NF, Y, L, I, H, R, E, K117W_I201C_A433C D, M, V. stabilizeprefusion axis interactions/ combined with DS 0187 A181BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, K117E, I201C/A433Cds/CavF/charged at trimer axis. T332N_ T332N Substitute-F, Y, L, I,K117E_I201C_A433C H, R, E, D, M, V. stabilize prefusion axisinteractions/combined with DS 0188 A182 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, K121E, I201C/A433C ds/CavF/charged at trimer axis.T332N_ T332N Substitute-F, Y, L, I, H, R, K121E_l201C_A433C E, D, M, V.stabilize prefusion axis interactions/combined with DS 0189 A183BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M426W, I201C/A433Cds/CavF at parallel b-strand #. T332N_ T332NSubstitute-F, Y, L, I, H, R, M426W_I201C_A433C E, D, V. stabilizeprefusion axis interactions/ combined with DS 0190 A184BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S110W, I201C/A433Cds/CavF at trimer axis/gp41 T332N_ T332N interface boundary. Substitute-S110W_I201C_A433C F, Y, L, I, M, V. stabilize prefusion axisinteractions/gp41 interface/prevent helix movement/combined with DS 0191A185 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q114W, I201C/A433CSame as Seq_0190 T332N_ T332N Q114W I201C A433C 0192 A186BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, P3220W, I201C/A433Cds/CavF at gp41 interface. T332N_ T332N Substitute-F, Y, L, I, M, V.P220W_I201C_A433C stabilize gp41 interface/ combined with DS 0193 A187BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T50W, I201C/A433Cds/CavF at gp41 interface. T332N_ T332N Substitute-F, Y, L, I, M, V.T50W_I201C_A433C stabilize gp41 interface/ combined with DS 0194 A188BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, R429W, I201C/A433Cds/CavF at parallel b-strand #. T332N_ T332NSubstitute-F, Y, L, I, M, V. R429W_I201C_A433C prevent bridging sheetformation/combined with DS 0195 A189 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Y61W CavF at Middle of α-1. T332N_ T332N Will fill aY61W cavity between gp120 and gp41 to revent CD4 induced α-1 disruptionand α0 formation 0196 A190 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,S209R, V68L CavF at Loop between α-1 and beta 0 T332N_ T332Nand loop between beta 3 and beta 4. S209R_V68LSubstitute-F, W, Y, L, I, M, V. Fills cavitiesthat are otherwise filled by CD4 induced α0 formation 0197 A191BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, :53W, Q246WCavF at gp120-gp41 interface, T332N_ T332N N-term F53W_Q246Wof beta-2, middle of beta-8, Substitute-246 F, W, Y, L, I, M, V.Will fill a cavity between gp120 and zp41 to stabilize gp41 disorderedregion and interaction between gp120 and gp41 0198 A192BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Y177W, I323FCavF at C-term of beta C, T332N_ T332N C-term of Y177W_I323Fbeta V3B. Substitute-323 F, Y, W. Fills cavity between V3 and gp120core to stabilize closed cap 0199 A193 BG505SOS.R6.664. BG505SOS, R6, 664, G41C/A541C DS-Fixes ground state, DS between T332N_ T332Ngp120 and gp41 G41C A541C 0200 A194 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, G41C/A541C S-Fixes ground state, DS between T332N_ T332Ngp120 and gp41 G41C A541C 0201 A195 BG505.R6.664. BG505 R6, 664,G41C/A541C DS, non SOSIP-Fixes T332N_ T332N ground state, DS G41CA541Cbetween gp120 and gp41 0202 A196 BG505, SOS.R6.664. BG505 SOS, R6, 664,547-GGPGGPGG-569 Prevent α6 to α7 transition T332N_ T332N547-GGPGGPGG-569 0203 A197 BG505, SOS.R6.664. BG505 SOS, R6, 664,547-GGGGPGGPG-569 Prevent α6 to α7 transition T332N_ T332N547-GGGGPGGPG-569 0204 A198 BG505, SOS.R6.664. BG505 SOS, R6, 664,547-GGGPGGG-569 Prevent α6 to α7 transition T332N_ T332N 547-GGGPGGG-5690205 A199 BG505, SOS.R6.664. BG505 SOS, R6, 664, 547-GGPGGGGPGG-569Prevent α6 to α7 transition T332N_ T332N 547-GGPGGGGPGG-569 0206 A200BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q551PPrevent α6 to α7 transition T332N_ T332N Q551P 0207 A201BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L556PPrevent α6 to α7 transition T332N_ T332N L556P 0208 A202BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, H564PPrevent α6 to α7 transition T332NH564P T332N 0209 A203BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L568PPrevent α6 to α7 transition T332NL 568P T332N 0210 B001 BG505, IP.664.BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N, 10 A.A. linkerno SOS; bC-10ln with I559P 0211 B002 BG505, IP.664. BG505 IP, 664,508(REKR)511 replaced Single chain; T332N_ T332N by A.A. linker, no SOS;bC-10ln_R166W R166W with I559P; with R166W 0212 B003 BG505, IP.664.BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 11 A.A. linkerno SOS; bC-11ln with I559P 0213 B004 BG505, IP.664. BG505 IP, 664,508(REKR)511 → Same as Seq_211 T332N_ T332N 11 A.A. linker, R166WbC-11ln R166W 0214 B005 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Single chain; T332N_ T332N 12 A.A. linker no SOS; bC-12ln with I559P0215 B006 BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Same as Seq_211T332N_ T332N 12 A.A. linker, R166W bC-12ln R166W 0216 B007BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N13 A.A. linker no SOS; bC-13ln with I559P 0217 B008 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Same as Seq_211 T332N_ T332N13 A.A. linker, R166W bC-13ln_R166W 0218 B009 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 14 A.A. linkerno SOS; bC-14ln with I559P 0219 B010 BG505, IP.664. BG505 IP, 664,508(REKR)511 → Same as Seq_211 T332N_ T332N 14 A.A. linker, R166WbC-14ln_R166W 0220 B011 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Single chain; T332N_ T332N 15 A.A. linker no SOS; bC-15ln with I559P0221 B012 BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Same as Seq_211T332N_ T332N 15 A.A. linker, R166W bC-15ln_R166W 0222 B013BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N1 A.A. linker no SOS; bC-11ln with I559P 0223 B014 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Same as Seq_211 T332N_ T332N1 A.A. linker, R166W bC-11ln_R166W 0224 B015 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 20 A.A. linkerno SOS; bC-20ln with I559P 0225 B016 BG505, IP.664. BG505 IP, 664,508(REKR)511 → Same as Seq_211 T332N_ T332N 20 A.A. linker, R166WbC-20ln_R166W 0226 B017 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Single chain; T332N_ T332N 2 A.A. linker no SOS; bC-2ln with I559P 0227B018 BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Same as Seq_211 T332N_T332N 2 A.A. linker, R166W bC-2ln_R166W 0228 B019 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 3 A.A. linker no SOS;bC-3ln with I559P 0229 B020 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Same as Seq_211 T332N_ T332N 3 A.A. linker, R166W bC-3ln_R166W 0230 B021BG505, IP.664. BG505 IP, 664, 508(REKR)511 replaced Single chain; T332N_T332N by 4 A.A. linker no SOS; bC-4ln with I559P 0231 B022BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Same as Seq_211 T332N_T332N 4 A.A. linker, R166W bC-4ln R166W 0232 B023 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 5 A.A. linker no SOS;bC-5ln with I559P 0233 B024 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Same as Seq_211 T332N_ T332N 5 A.A. linker, R166W bC-5ln_R166W 0234 B025BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N6 A.A. linker no SOS; bC-6ln with I559P 0235 B026 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Same as Seq_211 T332N_ T332N6 A.A. linker, R166W bC-6ln_R166W 0236 B027 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 7 A.A. linker no SOS;bC-7ln with I559P 0237 B028 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Same as Seq_211 T332N_ T332N 7 A.A. linker, R166W bC-7ln_R166W 0238 B029BG505, IP.664. BG505 IP, 664, 508(REKR)511 → Single chain; T332N_ T332N8 A.A. linker no SOS; bC-8ln with I559P 0239 B030 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Same as Seq_211 T332N_ T332N8 A.A. linker, R166W bC-8ln R166W 0240 B031 BG505, IP.664. BG505IP, 664, 508(REKR)511 → Single chain; T332N_ T332N 9 A.A. linker no SOS;bC-9ln with I559P 0241 B032 BG505, IP.664. BG505 IP, 664, 508(REKR)511 →Same as Seq_211 T332N_ T332N 9 A.A. linker, R166W bC-9ln R166W 0242 B033BG505.664. BG505 564, T332N 508(REKR)511 → Single chain; T332N_10 A.A. linker no SOSIP C-10ln 0243 B034 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 10 A.A. linker; no SOS;C-10ln_Q551F Q551F withQ551F 0244 B035 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 11 A.A. linker no SOSIP C-11ln0245 B036 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_T332N 12 A.A. linker no SOSIP C-12ln 0246 B037 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 13 A.A. linker no SOSIP C-13ln0247 B038 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_T332N 14 A.A. linker no SOSIP C-14ln 0248 B039 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 15 A.A. linker no SOSIP C-15ln0249 B040 BG505.664. BG505 564, T332N 508(REKR)511 → Single chain;T332N_ 1 A.A. linker no SOSIP C-1ln 0250 B041 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 2 A.A. linker no SOSIP C-2ln0251 B042 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_T332N 3 A.A. linker no SOSIP C-3ln 0252 B043 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 4 A.A. linker no SOSIP C-4ln0253 B044 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_T332N 5 A.A. linker no SOSIP C-5ln 0254 B045 BG505.664. BG505 664,508(REKR)511 → Single chain; T332N_ T332N 6 A.A. linker no SOSIP C-6ln0255 B046 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_T332N 7 A.A. linker no SOSIP C-7ln 0256 B047 BG505.664. BG505 564, T332N508(REKR)511 → Single chain; T332N_ 8 A.A. linker no SOSIP C-8ln 0257B048 BG505.664. BG505 664, 508(REKR)511 → Single chain; T332N_ T332N9 A.A. linker no SOSIP C-9ln 0258 B049 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N Gp120-HR2-HR1 0259 B050BG505.IP.664. BG505 IP, 664, linker gp120-HR1 T332N_ T332NGp120-Linker-HR1-GCN4 0260 B051 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit2noC 0261 B052BG505.IP.664. BG505 IP, 664, circular permutant single chain T332N_T332N SOScircuit3-noFus 0262 B053 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit4noC-noFus 0263B054 BG505.IP.664. BG505 IP, 664, circular permutant single chain T332N_T332N SOScircuit9-GCN4coredownCC 0264 B055 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit14-HR1envHR2-GCN4coredown 0265 B056 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit15-HR1envHR2-GCN4coredown 0266 B057 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit16-HR1envHR2-GCN4coredown 0267 B058 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit17-HR1envHR2 0268B059 BG505.IP.664. BG505 IP, 664, circular permutant single chain T332N_T332N SOScircuit18-HR1envHR2 0269 B060 BG505.IP.664. BG505 IP, 664,circular permutant single chain T332N_ T332N SOScircuit4noC-noFus_CMVR-3c-His-R166W 0270 B061 BG505.IP.664. BG505 IP, 664, R166W, circ. permut.circ. Permut. single chain in ZM53, T332N_ T332N with R166W 1.ZM53.R166W0271 B062 BG505.IP.664. BG505 IP, 664, R166W, circ. permut.circ. Permut. single chain in ZM53, T332N_ T332N with R166W10.ZM53.R166W 0272 B063 BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N_ T332N 10_BG505_Nterm_Hl 0273 B064BG505.IP.664. BG505 IP, 664, P313W, circ. permut.circular permutant single chain T332N_ T332N 10_BG505_Nterm_H1_P313W0274 B065 BG505.IP.664. BG505 IP, 664, P313W, R166W, circ.circular permutant single chain T332N_ T332N permut. -R166W_10_BG505_Nterm_H1_P313W 0275 B066 BG505.IP.664. BG505 IP, 664,R166W, circ. permut. circular permutant single chain T332N_ T332N10_BG505_Nterm_H1_R166W 0276 B067 BG505.IP.664. BG505 IP, 664,R166W, circ. permut. circular permutant single chain T332N_ T332N11.ZM53.R166W 0277 B068 BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N_ T332N 11_BG505_Nterm_Hl 0278 B069BG505.IP.664. BG505 IP, 664, P313W, circ. permut.circular permutant single chain T332N_ T332N 11_BG505_Nterm_Hl_P313W0279 B070 BG505.IP.664. BG505 IP, 664, P313W, R166W, circ.circular permutant single chain T332N.-R166W T332N permut._11_BG505_Nterm_H1_P313W 0280 B071 BG505.IP.664. BG505 IP, 664,R166W, circ. permut. circular permutant single chain T332N_ T332N11_BG505_Nterm_Hl_R166W 0281 B072 BG505.IP.664. BG505 IP, 664,R166W, circ. permut. circular permutant single chain T332N_ T332N12.ZM53.R166W 0282 B073 BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N_ T332N 12_BG505_Nterm_Hl 0283 B074BG505.IP.664. BG505 IP, 664, P313W, circ. permut.circular permutant single chain T332N_ T332N 12_BG505_Nterm_Hl_P313W0284 B075 BG505.IP.664. BG505 IP, 664, P313W, R166W, circ.circular permutant single chain T332N.-R166W T332N permut._12_BG505_Nterm_H1_P313W 0285 B076 BG505.IP.664. BG505 IP, 664,R166W, circ. permut. circular permutant single chain T332N_ T332N12_BG505_Nterm_H1_R166W 0286 B077 BG505.IP.664. BG505 IP, 664,Linker at cleavage site Linker at cleavage site T332N_ T332N13_BG505_6RLinked 0287 B078 BG505.IP.664. BG505 IP, 664,Linker at cleavage site Linker at cleavage site T332N_ T332N14_BG505_6RLinked 0288 B079 BG505.IP.664. BG505 IP, 664,Linker at cleavage site Linker at cleavage site T332N_ T332N15_BG505_6RLinked 0289 B080 BG505.IP.664. BG505 IP, 664,Linker at cleavage site Linker at cleavage site T332N_ T332N16_BG505_6RLinked 0290 B081 BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N_ T332N 1_BG505_Nterm_H1 0291 B082BG505.IP.664. BG505 IP, 664, R166W, circ. permut.circular permutant single chain T332N.__l_BG505_ T332N Nterm_H1_R166W0292 B083 BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N_ T332N 2_BG505_Nterm_H1 0293 B084BG505.IP.664. BG505 IP, 664, circ. permut.circular permutant single chain T332N.__3_BG505_Nterm_H1 T332N 0294 B085BG505.IP.664. BG505 IP, 664, R166W, circ. permut.circular permutant single chain in T332N_ T332N ZM53, with R166W4.ZM53.R166W 0295 B086 BG505.IP.664. BG505 IP, 664, circ. permut.Circular permutant single chain T332N_ T332N 4_BG505_Nterm_Hl 0296 B087BG505.IP.664. BG505 IP, 664, R166W, circ. permut.circular permutant single chain, T332N_ T332N with R166W4_BG505_Nterm_H1_R166W 0297 B088 BG505.IP.664. BG505 IP, 664,circ. permut. Circular permutant single chain T332N_ T332N5_BG505_Nterm_H1 0298 B089 BG505.IP.664. BG505 IP, 664, circ. permut.Circular permutant single chain T332N_ T332N 6_BG505_Nterm_H1 0299 B090BG505.IP.664. BG505 IP, 664, circ. permut.Circular permutant single chain T332N_ T332N 7_BG505_Nterm_H1 0300 B091BG505.IP.664. BG505 IP, 664, R166W circ. permut.circular permutant single chain, T332N_ T332N with R166W7_BG505_Nterm_H1_R166W 0301 B092 BG505.IP.664. BG505 IP, 664,circ. permut. Circular permutant single chain T332N_ T332N8_BG505_Nterm_H1 0302 B093 BG505.IP.664. BG505 IP, 664, circ. permut.Circular permutant single chain T332N_ T332N 9_BG505_Nterm_H1 0303 B094BG505.SOSIP.664. BG505 SOSIP, 664, A433P, circ. permut.circular permutant single chain, T332N_ T332N with A433P29_gp120-HR2_A433P 0304 B095 BG505.SOSIP.664. BG505 SOSIP, 664,A433P, circ. permut. circular permutant single chain, T332N_ T332Nwith A433P 30_gp120-HR2_A433P 0305 B096 CH117.4_332N_IP_10ln CH117.4 IP;508(REKR)511 → Single chain; 664; 10 A.A. linker; no SOS; T332N 332Nwith I559P 0306 B097 CNE58_IP_10ln CNE58 IP; 508(REKR)511 →Single chain; 664 10 A.A. linker no SOS; with I559P 0307 B098Cap256-SU_IP_10ln Cap256-SU IP; Same as Seq_307 Single chain; 664no SOS; with I559P 0308 B099 SHIV-1157ipd3N4_IP_10ln 1157ipd3N4 IP;Same as Seq_307 Single chain; 664 no SOS; with I559P 0309 B100ZM53_IP_10ln ZM53 IP; Same as Seq_307 Single chain; 664 no SOS;with I559P 0310 B101 C-10ln_Q551F BG505 664 664; Single chain;508(REKR)511 → no SOS; 10 A.A. linker; with I559P; Q551F with Q551F 0311B102 TF-B_THRO_TFl_10ln_Q551F THRO_TF1 664 664; Single chain;508(REKR)511 → no SOS; 10 A.A. linker; withQ551F Q551F 0312 B103TF-C_1245045_10ln_Q551F 1245045 664 664; Single chain; 508(REKR)511 →no SOS; 10 A.A. linker; withQ551F Q551F 0313 B104 3301_V1_C24_10ln_Q551F3301_V1_C24 664 664; Single chain; 508(REKR)511 → no SOS;10 A.A. linker; withQ551F Q551F 0314 B105 TF-C_19157834_v1_ 19157834_v1664 664; Single chain; 10ln_Q551F_P162T 508(REKR)511 → no SOS;10 A.A. linker; withQ551F 0551F; P162T 0315 B106 25925-2.22_10ln_Q551F25925-2.22 IP, 664 508(REKR)511 → Single chain; 10 A.A. linker; no SOS;Q551F withQ551F 0316 B107 CAP210.E8_10ln_Q551F CAP210.E8 IP, 664508(REKR)511 → Single chain; 10 A.A. linker; no SOS; Q551F withQ551F0317 B108 CNE58_IP_10ln_SU-strandC CNE58 IP, 664 664; Single chain;508(REKR)511 → no SOS; 10 A.A. linker with I559P;strand-C from CAP256-SU 0318 B109 CNE58_10ln_Q551F_ CNE58 IP, 664508(REKR)511 → Single chain; SU-strandC 10 A.A. linker; no SOS; 0551FwithQ551F; strand-C from CAP256-SU 0319 B110 TF-B_THRO_TF1_IP_10lnTHRO_TF1 IP, 664 Same as Seq_307 Single chain; no SOS; with I559P 0320B111 25925-2.22_IP_10ln 25925-2.22 IP, 664 508(REKR)511 → Single chain;10 A.A. linker no SOS; with I559P 0321 B112 CAP210.E8_IP_10ln CAP210.E8IP, 664 508(REKR)511 → Single chain; 10 A.A. linker no SOS; with I559P0322 B113 TF-C_19157834_v1_ 19157834_v1 IP, 664 664; Single chain;IP_10ln_P162T 508(REKR)511 → no SOS; 10 A.A. linker, with I559P P162T0323 B114 3301_V1_C24_IP_10ln 3301 V1 C24 IP, 664 664; Single chain;508(REKR)511 → no SOS; 10 A.A. linker with I559P 0324 B115TF-C_1245045_IP_10ln 1245045 IP, 664 664; Single chain; 508(REKR)511 →no SOS; 10 A.A. linker with I559P 0325 B116 00836-2.5_332N_IP_10ln00836-2.5 IP, 664 664; Single chain; 508(REKR)511 → no SOS;10 A.A. linker, with I559P 332N 0326 B117 6322_V4_C1_332N_IP_10ln6322_V4_C1 IP, 664 664; Single chain; 508(REKR)511 → no SOS;10 A.A. linker, with I559P 332N 0327 B118 ZM53-R166W_bC-10ln_IP ZM53IP, 664 664; Single chain; 508(REKR)511 → no SOS; 10 A.A. linker,with I559P R166W 0328 B119 ZM53-R166W_bC-15ln_IP ZM53 IP, 664 664;Single chain; 508(REKR)511 → no SOS; 15 A.A. linker, with I559P R166W0329 B120 ZM53-R166W_bC-20ln_IP ZM53 IP, 664 664; Single chain;508(REKR)511 → no SOS; 20 A.A. linker, with I559P R166W 0330 B121ZM53-R166W_bC-7ln_IP ZM53 IP, 664 664; Single chain; 508(REKR)511 →no SOS; 7 A.A. linker, with I559P R166W 0331 B122 ZM53-R166W_C-10lnBG505 664 508(REKR)511 → Single chain; 10 A.A. linker, R166W no SOS 0332B123 ZM53-R166W_C-15ln BG505 664 508(REKR)511 → Single chain;15 A.A. linker, R166W no SOS 0333 B124 ZM53-R166W_C-20ln BG505 664508(REKR)511 → Single chain; 20 A.A. linker, R166W no SOS 0334 B125ZM53-R166W_C-7ln BG505 664 508(REKR)511 → Single chain;7 A.A. linker, R166W no SOS 0335 B126 BG505.SOSIP.664. BG505 SOSIP, 664,504-518 → Single chain + N-term of gp41; T332N_ T332N 5 A.A. linker withdel504-518_5ln SOS; with I559P 0336 B127 BG505.SOSIP.664. BG505SOSIP, 664, 504-518 → Same as Seq_0335 T332N_ T332N 10 A.A. linkerdel504-518_10ln 0337 B128 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-518 → Same as Seq_0335 T332N_ T332N 15 A.A. linkerdel504-518_15ln 0338 B129 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-518 → Same as Seq_0335 T332N_ T332N 20 A.A. linkerdel504-518_20ln 0339 B130 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-518 → Same as Seq_0335 T332N_ T332N 5 A.A. linkerdel505-518_5ln 0340 B131 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-518 → Same as Seq_0335 T332N_ T332N 10 A.A. linkerdel505-518_10ln 0341 B132 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-518 → Same as Seq_0335 T332N_ T332N 15 A.A. linkerdel505-518_15ln 0342 B133 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-518 → Same as Seq_0335 T332N_ T332N 20 A.A. linkerdel505-518_20ln 0343 B134 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-521 → Same as Seq_0335 T332N_ T332N 5 A.A. linkerdel504-521_5ln 0344 B135 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-521 → Same as Seq_0335 T332N_ T332N 10 A.A. linkerdel504-521_10ln 0345 B136 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-521 → Same as Seq_0335 T332N_ T332N 15 A.A. linkerdel504-521_15ln 0346 B137 BG505.SOSIP.664. BG505 SOSIP, 664,segment 504-521 → Same as Seq_0335 T332N_ T332N 20 A.A. linkerdel504-521_20ln 0347 B138 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-521 → Same as Seq_0335 T332N_ T332N 5 A.A. linkerdel505-521_5ln 0348 B139 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-521 → Same as Seq_0335 T332N_ T332N 10 A.A. linkerdel505-521_10ln 0349 B140 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-521 → Same as Seq_0335 T332N_ T332N 15 A.A. linkerdel505-521_15ln 0350 B141 BG505.SOSIP.664. BG505 SOSIP, 664,segment 505-521 → Same as Seq_0335 T332N_ T332N 20 A.A. linkerdel505-521_20ln 0351 B142 BG505.SOSIP.664. BG505 SOSIP, 664,508(REKR)511 → Same as Seq_0335 T332N_ T332N 5 A.A. linker c5ln 0352B143 BG505.SOSIP.664. BG505 SOSIP, 664, 508(REKR)511 → Same as Seq_0335T332N_ T332N 10 A.A. linker c10ln 0353 B144 BG505.SOSIP.664. BG505SOSIP, 664, 508(REKR)511 → Same as Seq_0335 T332N_ T332N 15 A.A. linkercl5ln 0354 B145 BG505.SOSIP.664. BG505 SOSIP, 664, 508(REKR)511 →Same as Seq_0335 T332N_ T332N 20 A.A. linker c20ln 0355 B146BG505.IP.664. BG505 IP, 664, segment 505-521 →Single chain + N-term of gp41; T332N_ T332N 5 A.A. linker nodel505-521_5ln SOS; with I559P 0356 B147 BG505.IP.664. BG505 IP, 664,segment 505-521 → Single chain + N-term of gp41; T332N_ T332N10 A.A. linker no del505-521_10ln SOS; with I559P 0357 B148BG505.IP.664. BG505 IP, 664, segment 505-521 →Single chain+ N-term of gp41; T332N_ T332N 15 A.A. linker no SOS;del505-521_15ln with I559P 0358 B149 BG505.IP.664. BG505 IP, 664,segment 505-521 → Single chain+ N-term of gp41; T332N_ T332N20 A.A. linker no SOS; del505-521_20ln with I559P 0359 B150BG505.SOSIP.664. BG505 SOSIP, 664, cleavage site and NT Same as Seq_0335T332N_ T332N effusion peptide sc BZI replaced by a flexible linker 0360B151 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ2 0361 B152 BG505.SOSIP.664. BG505SOSIP, 664, Same as Seq_0359 Same as Seq_0335 T332N_ T332N sc BZ3 0362B153 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ4 0363 B154 BG505.SOSIP.664. BG505SOSIP, 664, Same as Seq_0359 Same as Seq_0335 T332N_ T332N sc BZ5 0364B155 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ6 0365 B156 BG505.SOSIP.664. BG505SOSIP, 664, Same as Seq_0359 Same as Seq_0335 T332N_ T332N sc BZ7 0366B157 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ8 0367 B158 BG505.SOSIP.664. BG505SOSIP, 664, Same as Seq_0359 Same as Seq_0335 T332N_ T332N sc BZ9 0368B159 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ10 0369 B160 BG505.SOSIP.664. BG505SOSIP, 664, Same as Seq_0359 Same as Seq_0335 T332N_ T332N sc BZ11 0370B161 BG505.SOSIP.664. BG505 SOSIP, 664, Same as Seq_0359Same as Seq_0335 T332N_ T332N sc BZ12 0371 B162 BG505.SOSIP.664. BG505SOSIP, 664, cleavage site and NT Same as Seq_0335 T332N_ T332Nof fusion peptide sc BZ2 replaced by a G312C/S199C/R166C/V127Cflexible linker; G312C/S199C/R166C/V127C 0372 B163 BG505.SOSIP.664.BG505 SOSIP, 664, cleavage site and NT Same as Seq_0335 T332N_ T332Nof fusion peptide sc BZ3 replaced by a G312C/S199C/R166C/V127Cflexible linker; G312C/S199C/R166C/V127C 0373 B164 BG505.IP. BG505IP, 664, on ferritin with ferritin for linker 664. a 5-linkerreplacement of T332N_ T332N gp120-gp41 cleavage site; bC10ln-5ln-no SOS; HpyFerritin with I559P 0374 B165 BG505.IP. BG505 IP, 664,on ferritin with Same as Seq_0373 664. a 10-1 inker T332N_ T332NbC10ln-10ln- HpyFerritin 0375 B166 BG505.IP.664. BG505 IP, 664,on ferritin with Same as Seq_0373 T332N_ T332N a 15-1 inker bC10ln-15ln-HpyFerritin 0376 B167 BG505.664. BG505 664, on ferritinferritin for linker T332N_ with a 5-linker; replacement ofFC10ln-5ln-HpyFerritin T332N Q551F gp120-gp41 cleavage site; no SOS;with Q551F 0377 B168 BG505.664. BG505 564, T332N on ferritin withSame as Seq_0376 T332N_ a 10-1 inker; FC10ln-10ln-HpyFerritin Q551F 0378B169 BG505.664. BG505 564, T332N on ferritin with Same as Seq_0376T332N_ a 15-1 inker; FC10ln-15ln-HpyFerritin Q551F 0379 H009Cap256-SU_bg505- Cap256-SU/BG505 SOSIP; 3G505 Platform, NCgp120 +chimera 664; remainder = Cap256-SU gp41.SOSIP R6 0380 H0101157ipd3N4_bg505- 1157ipd3N4/BG505 SOSIP; BG505 Platform,NCgp120 + gp41.SOSIP chimera 664; remainder = R6 1157ipd3N4 0381 H011CH117.4_332N_bg505- CH117.4_332N/BG505 SOSIP; 3G505 Platform,NCgp120 + gp41.SOSIP 664; remainder = R6 chimera CH117.4_332N 0382 H012CNE58_SU-strandC_bg505- CNE58_SU-strandC/ SOSIP; 3G505 Platform,NCgp120 + gp41.SOSIP BG505 664; Res. 166-173 (strand C) chimera R6From CAP256-SU, remainder = CNE58 0383 H013 25925-2.22_bg505-25925-2.22/BG505 SOSIP; 3G505 Platform, NCgp120 + gp41.SOSIP chimera664; remainder = 25925-2.22 R6 0384 H014 3301_V1_C24_bg505-3301_V1_C24/BG505 SOSIP; 3G505 Platform, NCgp120 + gp41.SOSIP chimera664; remainder = R6 3301 V1 C24 0385 H015 ZM53-R166W_bg505-ZM53-R166W/BG505 SOSIP; 3G505 Platform, NCgp120 + gp41.SOSIP chimera664; remainder = ZM53- R6 R166W 0386 H016 ZM53_bg505- ZM53/BG505 SOSIP;3G505 Platform, NCgp120 + gp41.SOSIP chimera 664; remainder = ZM53 R6SP120 0387 B170 BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutantLink gp120 Cterm T332N_ T332N to gp41 resi 664, SC_1 and circ. Permutatethru rest of gp41, end at 518 0388 B171 BG505.SOSIP.664. BG505SOSIP, 664, Circular permutant same as PA SC 1 T332N_ T332Nabove, vary linker SC_2 0389 B172 BG505.SOSIP.664. BG505 SOSIP, 664,Circular permutant same as PA_SC_1 and PA_SC_2 T332N_ T332Nabove, vary linker SC_3 0390 B173 BG505.SOSIP.664. BG505 SOSIP, 664,Circular permutant connects gp120 C term to region T332N_ T332Naround 606-609, and extends SC_4 Forward, turncating at differentregions) 0391 B174 BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutantonly the innercircle of T332N_ T332N helices to hold SC_5the trimer together 0392 B175 BG505.SOSIP.664. BG505 SOSIP, 664,Circular permutant Same as Seq_0391 T332N_ T332N SC_6 0393 B176BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutant Same as Seq_0391T332N_ T332N SC_7 0394 B177 BG505.SOSIP.664. BG505 SOSIP, 664,Circular permutant Same as Seq_0391 T332N_ T332N SC_8 0395 B178BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutantStart from resi 518, go to 664, T332N_ T332Nconnect to C-term of gp120, circ. SC_9 permutate thru gp120 0396 B179BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutantsame as PA SC 9, change linker T332N_ T332N SC_10 0397 B180BG505.SOSIP.664. BG505 SOSIP, 664, Circular permutantsame as PA SC 9, change linker T332N_ T332N SC_11 0398 B181BG505.IP.664. BG505 SOSIP, 664, Circular permutantCircular permutant single chain T332N_ T332N 1.2 0399 B182 BG505.IP.664.BG505 SOSIP, 664, Circular permutant Circular permutant single chainT332N_ T332N 1.3 0400 B183 BG505.IP.664. BG505 SOSIP, 664,Circular permutant Circular permutant single chain T332N_ T332N 2.1 0401B184 BG505.IP.664. BG505 SOSIP, 664, Circular permutantCircular permutant single chain T332N_ T332N 2.2 0402 B185 BG505.IP.664.BG505 SOSIP, 664, Circular permutant Circular permutant single chainT332N_ T332N 2.3 0403 B186 BG505.IP.664. BG505 SOSIP, 664,Circular permutant Circular permutant T332N_ T332N single chain 3.1 0404B187 BG505.IP.664. BG505 SOSIP, 664, Circular permutantCircular permutant T332N_ T332N single chain 3.3 0405 B188 BG505.IP.664.BG505 IP, 664, 44-500-gsg-34-43- Circular permutant T332N.scl T332Ngsgg-526-664 single chain 0406 B189 BG505.SOSIP.664. BG505 SOSIP, 664,44-502-ggsgg-34-43- Circular permutant T332N.sc2 T332N gsgg-526-664single chain 0407 B190 BG505.IP.664. BG505 IP, 664, 44-500-gsg-34-42-Circular permutant gsgg-525-664 single chain T332N.sc3 T332N 0408 B191BG505.SOSIP.664. BG505 SOSIP, 664, 44-502-ggsgg-34- Circular permutant42-gsgg-525-664 single chain T332N.sc4 T332N 0409 C001BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T90C gp140-35O22complex-T332N_ T332N disulfide to T90 35O22_S80 0410 C002 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, P238C gp140-35O22complex- T332N_ disulfide to P238T332N 35O22_P77 0411 C003 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,F529C gp140-35O22complex- T332_NT529 disulfide to T332N 35O22_D111 0412C004 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, D624C gp140-35O22complex-T332N_ disulfide to D624 T332N 35O22_L109 or G112 0413 C005BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V625C gp140-35O22complex-T332N_N625 disulfide to T332N 35O22_L109 0414 C006 35O22_P77C Ab 35O22P77C gp140-35O22complex- 3G505.SOSIP.R6.664. T332N_ P238 0415 C00735O22_S80C Ab 35O22 S80C gp140-35O22complex- 3G505.SOSIP.R6.664. T332N_T90 0416 C008 35O22_L109C Ab 35O22 L109C gp140-35O22complex-BG505.SOSIP.R6.664. T332N_ D624 or BG505.SOSIP.R6.664. T332N_ N625 0417C009 35O22_D111C Ab 35O22 D111C gp140-35O22complex- 3G505.SOSIP.R6.664.T332N_ T529 0418 C010 35O22_G112C Ab 35O22 G112C gp140-35O22complex-3G505.SOSIP.R6.664. T332N_ D624 0419 C011 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, D624C gp140-35O22complex T332N_ T332N D624C 0420 C012BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, G459C gp140-VRC01complexT332N_ T332N G459C 0421 C013 JRFL IP 3C Strep G459C JRFL IP3C Strep G459C gp140-VRC01complex 0422 C014 BS208.B1 SOSIP, R6, 664,BS208.B1 SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0423 C015KER2018.11 SOSIP, R6, 664, KER2018.11 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0424 C016 C4118.09 SOSIP, R6, 664, C4118.09SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0425 C017TH966.8 SOSIP, R6, 664, TH966.8 SOSIP, R6, 664 G459C gp140-VRC01complexG459C 0426 C018 WIT0.33 SOSIP, R6, 664, WITO.33 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0427 C019 CH181.12 SOSIP, R6, 664, CH181.12SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0428 C020BB201.B42 SOSIP, R6, 664, BB201.B42 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0429 C021 Q842.dl2 SOSIP, R6, 664, Q842.dl2SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0430 C022AC10.29 SOSIP, R6, 664, AC10.29 SOSIP, R6, 664 G459C gp140-VRC01complexG459C 0431 C023 BX08 16SOSIP, R6, 664, BX08_16 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0432 C024 257102.43 SOSIP, R6, 664, 257102.43SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0433 C025259252.22 SOSIP, R6, 664, 259252.22 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0434 C026 SO18_18 SOSIP, R6, 664, SO18_18SOSIP, R6, 664 G459C gp140-VRC01complex G459C 0435 C027X1193.C1 SOSIP, R6, 664, X1193.C1 SOSIP, R6, 664 G459Cgp140-VRC01complex G459C 0436 C028 SU SOSIP, R6, 664, SU SOSIP, R6, 664G459C gp140-VRC01complex G459C 0437 C029 BG505.SOSIP G459C BG505 SOSIPG459C, V1V2 Swap BB201.B42 gp140-VRC01complex V1V2 Swap BB201.B42 0438C030 BG505.SOSIP G459C BG505 SOSIP G459C, V1V2 Swap KER2018.11gp140-VRC01complex V1V2 Swap KER2018.11 0439 C031 BG505.SOSIP G459CBG505 SOSIP G459C, V1V2 Swap CH070.1 gp140-VRC01complexV1V2 Swap CH070.1 0440 C032 BG505.SOSIP G459C BG505 SOSIPG459C, V1V2 Swap ZM233.6 gp140-VRC01complex V1V2 Swap ZM233.6 0441 C033BG505.SOSIP G459C BG505 SOSIP G459C, V1V2 Swap Q23.17 gp140-VRC01complexV1V2 Swap Q23.17 0442 C034 BG505.SOSIP G459C BG505 SOSIPG459C, V1V2 SwapA244 gp140-VRC01complex V1V2 Swap A244 0443 C035BG505.SOSIP G459C BG505 SOSIP G459C, V1V2 Swap WITO.33gp140-VRC01complex V1V2 Swap WIT0.33 0444 C036 BG505 Cap256G459CSU BG505SOSIP G459C, SU V1V2 swap gp140-VRC01complex V1V2 swap 0445 C037VRC01 HA60C-His VRC01 H Ab A60C 0446 C038 VRC01 H R61C-His VRC01 H AbA61C 0447 C039 VRC01 L VRC01L Ab 0448 C040 VRC01 H-His VRC01 H Ab 0449C041 VRC01LH scFvTbn-His-Strep VRC01 Ab GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS0450 C042 VRC01LH scFvTbn-His VRC01 Ab GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS0451 C043 VRC01LH scFv C60Tbn-His VRC01 Ab GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 0452 C044 simVRC01.2 (Thr) H VRC01 H Ab 0453 C045simVRC01.2 C60 (Thr) H VRC01 H Ab A60C 0454 C046 simVRC01.2 C60 (Thr) HSVRC01 H Ab A60C 0455 C047 simVRC01.2 L VRC01L Ab 0456 C048simVRC01.2 C60 (Thr) H VRC01 H Ab A60C 0457 C049 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, I323C covalently bonded gp140- T332N_ T332NPGT122complex I323C 0458 C050 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,G324C covalently bonded gp140- T332NG324C T332N PGT122complex 0459 C051G122LH F67C PGT122 H Ab F67C 0460 C052 G122LH G29C PGT122 H Ab G29C 0461D001 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 504N/506T Glycan at R504T332N_ T332N Glyc504 0462 D002 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,561N/663T Glycan at L661 T332N_ T332N Glyc661 0463 D003BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 504N/506T, 661N/663TGlycans at R504 and L661 T332N_ T332N Glyc504-661 0464 D004BG505, SOSIP.R6.664. BG505 SOSIP, R6, 664, K502N/R504T Glycan at K502T332N_ T332N K502N R504T 0465 D005 BG505, SOSIP.R6.664. BG505SOSIP, R6, 664, Q658N/L660T Glycan at Q658 T332N_ T332N Q658N L660T 0466D006 BG505, SOSIP.R6.664. BG505 SOSIP, R6, 664, W35T Glycan at N33T332N_ T332N W35T 0467 D007 BG505, SOSIP.R6.664. BG505 SOSIP, R6, 664,W35N Glycan at W35 T332N_ T332N W35N 0468 D008 BG505, SOSIP.R6.664.BG505 SOSIP, R6, 664, W35N, R504N/V506T Glycan at W35 and R504 T332N_T332N W35N R504N V506T 0469 D009 BG505, SOSIP.R6.664. BG505SOSIP, R6, 664, W35T, gly661 Glycan at N33 and L661 T332N_ T332NW35T_gly661 0470 D010 BG505, BG505 SOSIP, R6, 664,W35T, K502N/R504T, gly661 Glycan at K502 and L661 SOSIP.R6.664. T332NT332N_ W35T_K502N_R504T_gly661 0471 F001 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, onto ferritin HIV-1 En trimer on nanoparticles T332N_T332N ferritin 0472 F002 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,onto Luminase synthase HIV-1 En trimer on nanoparticles T332N_ T332N LS0473 F003 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Helicobacter pyloriSOSIP-linker-particle T332N_ T332N ferritin 3bve_ferr-24_1ln 0474 F004BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Helicobacter pyloriSOSIP-linker-particle T332N_ T332N ferritin 3bve_ferr-24_3ln 0475 F005BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Helicobacter pyloriSOSIP-linker-particle T332N_ T332N ferritin 3bve_ferr-24_15ln 0476 F006BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, lumazine synthase fromSOSIP-linker-particle T332N_ T332N aquifex aeolicus 1hqk_ls-60_3ln 0477F007 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, lumazine synthase fromSOSIP-linker-particle T332N_ T332N aquifex aeolicus 1hqk_ls-60_15ln 0478F008 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, DSDNA bacteriophageSOSIP-linker-particle T332N_ T332N HK97 mature lohg_bph-hk97-empty capsid 420_1ln 0479 F009 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,DSDNA bacteriophage SOSIP-linker-particle T332N_ T332N HK97 maturelohg_bph-hk97- empty capsid 420_3ln 0480 F010 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, DSDNA bacteriophage SOSIP-linker-particle T332N_ T332NHK97 mature lohg_bph-hk97- empty capsid 420_5ln 0481 F011BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, DSDNA bacteriophageSOSIP-linker-particle T332N_ T332N HK97 mature lohg_bph-hk97- 420_15ln0482 F012 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE Q BETASOSIP-linker-particle T332N_ T332N CAPSID iqbe_bph-qB-180_7ln 0483 F013BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE Q BETASOSIP-linker-particle T332N_ T332N CAPSID lqbe_bph-qB-180_15ln 0484 F014BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, bacteriophage PP7fromSOSIP-linker-particle T332N_ T332N Pseudomonas aeruginosa ldwn_bph-pp7-180_10ln 0485 F015 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,bacteriophage PP7from SOSIP-linker-particle T332N_ T332NPseudomonas aeruginosa ldwn_bph-pp7- 180_15ln 0486 F016BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, bacteriophage PRR1SOSIP-linker-particle T332N_ T332N 2vf9_bph-prr1- 180_101_n 0487 F017BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, bacteriophage PRR2SOSIP-linker-particle T332N_ T332N 2vf9_bph-prr1- 180_15ln 0488 F018BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE GASOSIP-linker-particle T332N_ T332N PROTEIN CAPSID Igav_bph-gα-180_10ln0489 F019 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE GASOSIP-linker-particle T332N_ T332N PROTEIN CAPSID Igav_bph-gα-180_15ln0490 F020 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,caulobacter bacteriophage SOSIP-linker-particle T332N_ T332N5-virus-like 2w4y_bph-5-180_10ln particle 0491 F021 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, caulobacter bacteriophage SOSIP-linker-particleT332N_ T332N 5-virus-like 2w4y_bph-5-180_15ln particle 0492 F022BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE FR CAPSIDSOSIP-linker-particle T332N_ T332N lfrs_bph-fr-180_10ln 0493 F023BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, BACTERIOPHAGE FR CAPSIDSOSIP-linker-particle T332N_ T332N lfrs_bph-fr-180_15ln 0494 F024BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, PHAGE MS2 PROTEIN CAPSIDSOSIP-linker-particle T332N_ T332N lmva_ph-ms2-180_7ln 0495 F025BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, PHAGE MS2 PROTEIN CAPSIDSOSIP-linker-particle T332N_ T332N lmva_ph-ms2- 180_15ln 0496 F026BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, tomato bushy stunt virus,particle-linker-SOSIP T332N_ T332N v. coat protein 2tbv_tom-v-180_7ln0497 F027 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,tomato bushy stunt virus, particle-linker-SOSIP T332N_ T332Nv. coat protein 2tbv_tom-v-180_15ln 0498 F028 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, SESBANIA MOSAIC VIRUS particle-linker-SOSIP T332N_ T332NCOAT PROTEIN lsmv_sesb-mv-180_7ln 0499 F029 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, SESBANIA MOSAIC VIRUS particle-linker-SOSIP T332N_ T332NCOAT PROTEIN lsmv_sesb-mv- 180_15ln 0500 F030 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, RICE YELLOW MOTTLE VIRUS particle-linker-SOSIP T332N_T332N lf2n_rice-ymv-180_7ln 0501 F031 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, RICE YELLOW MOTTLE VIRUS particle-linker-SOSIP T332N_T332N lf2n_rice-ymv- 180_15ln 0502 F032 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Cocksfoot mottle virus particle-linker-SOSIP T332N_T332N IngO_cfmv-180_7ln 0503 F033 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Cocksfoot mottle virus particle-linker-SOSIP T332N_T332N IngO_cfmv-180_15ln 0504 F034 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, protein cage by particle-linker-SOSIP T332N_ T332NEscherichia coli 2- 2wqt_mhpd-60_1ln hydroxypentadienoic acid hydratase0505 F035 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Same as Seq_0504particle-linker-SOSIP T332N_ T332N 2wqt_mhpd-60_3ln 0506 F036BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Same as Seq_0504particle-linker-SOSIP T332N_ T332N 2wqt_mhpd-60_5ln 0507 F037BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Same as Seq_0504particle-linker-SOSIP T332N_ T332N 2wqt_mhpd-60_15ln 0508 G001BG505.SOSIP.R6.664. BG505 SOSIP.R6.664. GGSGG GCN4 Trimerization domainT332N_ T332N GGSGG GCN4 0509 G002 BG505.SOSIP.R6.664. BG505SOSIP.R6.664. GGSGSGG N 3HSH Trimerization domain T332N_ T332Natgp120 N-term GGSGSGG N 3HSH 0510 G003 BG505.SOSIP.R6.664. BG505SOSIP.R6.664. GG C 3HSH Trimerization domain T332N_ T332N at gp41 C-termGG C 3HSH 0511 H001 BG505.SOSIP.R6.664. BG505 5OSIP.R6.664.V1V2 Swap CAP256.SU V1V2 Swap CAP256.SU T332N, V1V2 Swap CAP256.SU T332N0512 H002 BG505.SOSIP.R6.664. BG505 SOSIP.R6.664. V1V2 Swap BB201.B42V1V2 Swap BB201.B42 T332N, V1V2 Swap BB201.B42 T332N 0513 H003BG505.SOSIP.R6.664. BG505 5OSIP.R6.664. V1V2 Swap KER2018.11V1V2 Swap KER2018.11 T332N, V1V2 Swap KER2018.11 T332N 0514 H004BG505.SOSIP.R6.664. BG505 SOSIP.R6.664. V1V2 Swap CH070.1V1V2 Swap CH070.1 T332N, V1V2 Swap CH070.1 T332N 0515 H005BG505.SOSIP.R6.664. BG505 5OSIP.R6.664. V1V2 Swap ZM233.6V1V2 Swap ZM233.6 T332N, V1V2 Swap ZM233.6 T332N 0516 H006BG505.SOSIP.R6.664. BG505 SOSIP.R6.664. V1V2 Swap Q23.17V1V2 Swap Q23.17 T332N, V1V2 Swap Q23.17 T332N 0517 H007BG505.SOSIP.R6.664. BG505 SOSIP.R6.664. V1V2 Swap A244 V1V2 Swap A244T332N, V1V2 Swap A244 T332N 0518 H008 BG505.SOSIP.R6.664. BG505SOSIP.R6.664. V1V2 Swap WITO.33 V1V2 Swap WITO.33T332N, V1V2 Swap WITO.33 T332N 0519 Z001 0520 Z002 0521 Z003 0522 Z0040523 Z005 0524 Z006 0525 Z007 0526 Z008 0527 Z009 0528 Z010 0529 Z0110530 Z012 0531 Z013 0532 Z014 0533 Z015 0534 Z016 0535 Z017 0536 Z0180537 Z019 0538 Z020 0539 Z021 0540 Z022 0541 Z023 0542 Z024 0543 T001BG505SOSIP.R6.664. BG505 SOSIP.R6.664. Transmembrane T332N_ T332NC-6ln-HATM 0544 T002 BG505SOSIP.R6.664. BG505 SOSIP.R6.664.Transmembrane T332N_ T332N C-10ln-HATM 0545 T003 bC-10ln_IP-6ln-HATMBG505 Transmembrane 0546 T004 bC-10ln_IP-10ln-HATM BG505 Transmembrane0547 T005 BG505SOSIP.R6.664. BG505 SOSIP.R6.664. Transmembrane T332N_T332N N-NATM-6ln-C-6ln- HATM 0548 T006 BG505SOSIP.R6.664. BG505SOSIP.R6.664. Transmembrane T332N_ T332N C-10ln-HATM 0549 T007bC-10ln_IP-N-NATM- BG505 Transmembrane 6ln-6ln-HATM 0550 T008bC-10ln_IP-10ln-HATM BG505 Transmembrane 0551 T009 BG505SOSIP.R6.664.BG505 SOSIP.R6.664. A201C/A433C Transmembrane T332N_ T332NA201C/A433C-N-NATM- 6ln-C-6ln-HATM 0552 T010 BG505SOSIP.R6.664. BG5055OSIP.R6.664. A201C/A433C Transmembrane T332N_ T332N A201C/A433C-C-10ln-HATM 0553 T011 bC-10ln_IP-A201C/A433C- BG505 TransmembraneN-NATM-6ln-C-6ln-HATM 0554 T012 bC-10ln_IPA201C/A433C BG505Transmembrane 10ln-HATM 0555 T013 BG505SOSIP.R6.664. BG505 SOSIP.R6.664.A201C/A433C Transmembrane T332N_ T332N A201C/A433C-C-6ln- HATM 0556 T014BG505SOSIP.R6.664. BG505 SOSIP.R6.664. A201C/A433C Transmembrane T332N_T332N A201C/A433C-C-10ln- HATM 0557 T015 bC-10ln_IP-A201C/ BG505Transmembrane A433C-6ln-HATM 0558 T016 bC-10ln_IP-A201C/ BG505Transmembrane A433C-10ln-HATM 0559 T017 bC-10ln_IPA201C/A433C- BG505Transmembrane N-NATM-6ln-C-6ln-HATM 0560 T018 BG505SOSIP.R6.664. BG505SOSIP.R6.664. Transmembrane T332N_ T332N N-NATM-6ln 0561 T019BG505SOSIP.R6.664. BG505 SOSIP.R6.664. Transmembrane T332N_ T332NN-10ln-NATM 0562 T020 bC-10ln_IP-6ln-N-NATM BG505 Transmembrane 0563T021 bC-10ln_IP-10ln-N-NATM BG505 Transmembrane 0564 T022BG505SOSIP.R6.664. BG505 SOSIP.R6.664. A433P Transmembrane T332N_ T332NA433P-N-NATM-6ln-C- 6ln-HATM 0565 T023 BG505SOSIP.R6.664. BG505SOSIP.R6.664. A433P Transmembrane T332N_ T332N A433P-C-10ln-HATM 0566T024 bC-10ln_IPA433P-N- BG505 Transmembrane NATM-6ln-6ln-HATM 0567 T025bC-10ln_IPA433P 10ln-HATM BG505 Transmembrane 0568 T026BG505SOSIP.R6.664. BG505 SOSIP.R6.664. A433P Transmembrane T332N_ T332NA433P-C-6ln-HATM 0569 T027 BG505SOSIP.R6.664. BG505 SOSIP.R6.664. A433PTransmembrane T332N_ T332N A433P-C-10ln-HATM 0570 T028bC-10ln_IPA433P--6ln-HATM BG505 Transmembrane 0571 T029bC-10ln_IPA433P 10ln-HATM BG505 Transmembrane 0572 Z025 0573 Z026 0574Z027 0575 Z028 Ferritin subunit 0576 Z029 lumazine synthase subunit 0577Z030 Sulfer Oxygenase Reductase subunit 0578 Z031 Foldon domain 0579H017 Cap256-SU_bg505- CAP256-SU/BG505 SOSIP, R6, 664 BG505 PlatformChimeric gp140 with BG505 NCgp120 + chimera (Res. 31-45, 478-507,zp4lecto/gp120-NC+ Interface Res. int + gp41.SOSIP 512-664), BG505set A “platform” and heterologous Interface gpl20 (Int.) Res. setft (Res. 46-54; 70-75; 84-89; 99; 102; 106; 107; 114; 215;220-224; 226; 244; 471- 473; 476-477), remainder = Cap256-SU 0580 H018SHIV-1157ipd3N4_bg505- 5HIV-1157ipd3N4/BG505 SOSIP, R6, 664BG505 Platform, Same as Seq_0579 NCgp120 + chimeraBG505 Int. Res. set A; int + gp41.SOSIP remainder- SHIV-1157ipd3N4 0581H019 CH117.4_332N_bg505- CH117.4/BG505 SOSIP, R6, 664 BG505 Platform,Same as Seq_0579 NCgp120 + chimera BG505 Int. Res. set A;int + gp41.SOSIP remainder-CH117.4 gp120 0582 H020CNE58_SU-strandC_bg505- CNE58_SU-strandC/ SOSIP, R6, 664 BG505 Platform,Same as Seq_0579 NCgp120 + BG505 BG505 Int. Res. set A; int + gp41.SOSIPchimera remainder-CNE58 SU-strand C 0583 H021 25925-2.22_bg505-25925-2.22/BG505 SOSIP, R6, 664 BG505 Platform, Same as Seq_0579NCgp120 + chimera BG505 Int. Res. set A; int + gp41.SOSIPremainder-25925-2.22 0584 H022 3301_V1_C24_bg505- 3301_V1_C24/BG505SOSIP, R6, 664 BG505 Platform, Same as Seq_0579 NCgp120 + chimeraBG505 Int. Res. set A; int + gp41.SOSIP remainder-3301 VI C24 0585 H023ZM53-R166W_bg505- ZM53/BG505 SOSIP, R6, 664 BG505 Platform,Same as Seq_0579 NCgp120 + chimera BG505 Int. Res. set A;int + gp41.SOSIP remainder- ZM53-R166Wgp120 0586 H024 ZM53_bg505-ZM53/BG505 SOSIP, R6, 664 BG505 Platform, Same as Seq_0579 NCgp120 +chimera BG505 Int. Res. set A; int + gp41.SOSIP remainder-ZM53 0587 H025KER2018.11_bg505- KER2018.11/BG505 SOSIP, R6, 664 BG505 Platform,heterologous gp120 NCgp120 + gp41.SOSIP chimera remainder- with (gp41 +KER2018.11 gp120-NC (Res. 31-45; 478-507) from 3G505.SOSIP) 0588 H026KER2018.11_bg505- KER2018.11/BG505 SOSIP, R6, 664 BG505 Platform,Same as Seq_0579 NCgp120 + chimera BG505 Int. Res. set A;int + gp41.SOSIP remainder-KER2018.il 0589 H027 ZM233.6_bg505-ZM233.6/BG505 SOSIP, R6, 664 BG505 Platform, Same as Seq_0379NCgp120 + gp41.SOSIP chimera remainder-ZM233.6 0590 H028 ZM233.6_bg505-ZM233.6/BG505 SOSIP, R6, 664 BG505 Platform, Same as Seq_0579 NCgp120 +chimera BG505 Int. Res. set A; int + gp41.SOSIP remainder-ZM233.6 0591H029 UG037.8_bg505- UG037.8/BG505 SOSIP, R6, 664 BG505 Platform,Same as Seq_0379 NCgp120 + gp41.SOSIP chimera BG505 Int. Res. set A;remainder-UG037.8 0592 H030 C13_psv02_bg505- C13/BG505 SOSIP, R6, 664BG505 Platform, Same as Seq_0379 NCgp120 + gp41.SOSIP chimeraBG505 Int. Res. set A; remainder-C13 0593 H031 45_01dG5_bg505-45_01dG5/BG505 SOSIP, R6, 664 BG505 Platform, Same as Seq_0379NCgp120 + gp41.SOSIP chimera BG505 Int. Res. set A; remainder-45 01dG50594 H032 ZM215.8-dCD4bsGlyc_bg505- ZM215.8/BG505 SOSIP, R6, 664BG505 Platform, Same as Seq_0379 NCgp120 + gp41.SOSIP chimeraBG505 Int. Res. set A; remainder-ZM215.8- dCD4bsGlyc 0595 H033426c-dCD4bsGlyc_bg505- 426c/BG505 SOSIP, R6, 664 BG505 Platform,Same as Seq_0379 NCgp120 + gp41. chimera BG505 Int. Res. set A; SOSIPremainder- 426c-dCD4bsGlyc 0596 F038 3g505.sosip_ BG505 SOSIP, R6, 664,bacteriophage PP7 sosip-linker-particle ldwn_bph-pp7-180_3-127_Sin_mut1T332N 0597 F039 bg505.sosip_ BG505 SOSIP, R6, 664, bacteriophage PP7Same as Seq_0596 ldwn_bph-pp7-180_3-127_8ln_mut1 T332N 0598 F040g505.sosip_ BG505 SOSIP, R6, 664, bacteriophage PP7 Same as Seq_0596ldwn_bph-pp7-180_3-127_8ln_mut1 T332N 0599 F041 bg505.sosip_ BG505SOSIP, R6, 664, BACTERIOPHAGE Q BETA Same as Seq_0596lqbe_bph-qB-180_6-132_3ln_mut1 T332N CAPSID 0600 F042 bg505.sosip_ BG505SOSIP, R6, 664, BACTERIOPHAGE Q BETA Same as Seq_0596lqbe_bph-qB-180_6-132_Sin_mut1 T332N CAPSID 0601 F043 bg505.sosip_ BG505SOSIP, R6, 664, BACTERIOPHAGE Q BETA Same as Seq_0596lqbe_bph-qB-180_6-132_10ln_mut1 T332N CAPSID 0602 F044 bg505.sosip_BG505 SOSIP, R6, 664, bacteriophage PRR1 Same as Seq_05962vf9_bph-prr1-180_6-131-3ln_mut1 T332N 0603 F045 bg505.sosip_ BG505SOSIP, R6, 664, bacteriophage PRR1 Same as Seq_05962vf9_bph-prr1-180_6-131-5ln_mut1 T332N 0604 F046 bg505.sosip_ BG505SOSIP, R6, 664, bacteriophage PRR1 Same as Seq_0596 2vf9_bph-prr1- T332N180_6-131-10ln_mut1 0605 F047 3g505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE GA Same as Seq_0596 lgav_bph-gα- T332N PROTEIN CAPSID180_7-129_3ln_mut1 0606 F048 bg505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE GA PROTEIN Same as Seq_0596 lgav_bph-gα- T332N CAPSID180_7-129_5ln_mut1 0607 F049 3g505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE GA PROTEIN Same as Seq_0596 lgav_bph-gα- T332N CAPSID180_7-129_10ln_mut1 0608 F050 bg505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE FR CAPSID Same as Seq_0596 lfrs_bph-fr- T332N180_7-129_5ln_mut1 0609 F051 sg505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE FR CAPSID Same as Seq_0596 lfrs_bph-fr- T332N180_7-129_10ln_mut1 0610 F052 bg505.sosip_ BG505 SOSIP, R6, 664,BACTERIOPHAGE FR CAPSID Same as Seq_0596 lfrs_bph-fr-180_ T332N7-129_15ln_mut1 0611 F053 3g505.sosip_ BG505 SOSIP, R6, 664,PHAGE MS2 PROTEIN CAPSID Same as Seq_0596 lmva_ph-ms2-180_ T332N7-129_Sin_mut1 0612 F054 bg505.sosip_ BG505 SOSIP, R6, 664,PHAGE MS2 PROTEIN CAPSID Same as Seq_0596 lmva_ph-ms2-180_ T332N7-129_10ln_mut1 0613 F055 3g505.sosip_ BG505 SOSIP, R6, 664,PHAGE MS2 PROTEIN CAPSID Same as Seq_0596 lmva_ph-ms2-180_ T332N7-129_15ln_mut1 0614 F056 bg505.sosip_ BG505 SOSIP, R6, 664,LUMAZINE SYNTHASE Same as Seq_0596 1hqk_ls-60_9ln_mut1 T332NFROM AQUIFEX AEOLICUS 0615 F057 bg505.sosip_ BG505 SOSIP, R6, 664,Helicobacter pylori Same as Seq_0596 3bve_ferr-24_9ln_mut1 T332Nferritin 0616 F058 BG505.SOSIP. BG505 SOSIP, R6, 664, M1 proteinsosip-self- linker_1AA7_Ln9 T332N assembling protein cage 0617 F059BG505.SOSIP. BG505 SOSIP, R6, 664, M1 protein sosip-self-linker_1AA7_Lnl2 T332N assembling protein cage 0618 F060 BG505.SOSIP.BG505 SOSIP, R6, 664, M1 protein sosip-self- inker_1AA7_Lnl5 T332Nassembling protein cage 0619 F061 BG505.SOSIP. BG505 SOSIP, R6, 664,M1 protein sosip-self- linker_1AA7_Lnl8 T332N assembling protein cage0620 F062 BG505SOSIP- BG505 SOSIP, R6, 664, 3VDX Cage sosip-self-linker3-3VDX T332N assembling protein cage 0621 F063 BG505SOSIP- BG505SOSIP, R6, 664, 3VDX Cage sosip-self- linker8-3VDX T332N assemblingprotein cage 0622 F064 BG505SOSIP-linker9-3VDX BG505 SOSIP, R6, 664,3VDX Cage sosip-self- T332N assembling protein cage 0623 F065BG505SOSIP-linkerl2-3VDX BG505 SOSIP, R6, 664, 3VDX Cage sosip-self-T332N assembling protein cage 0624 F066 BG505SOSIP-linkerl5-3VDX BG505SOSIP, R6, 664, 3VDX Cage sosip-self- T332N assembling protein cage 0625F067 BG505SOSIP-linkerl8-3VDX BG505 SOSIP, R6, 664, 3VDX Cagesosip-self- T332N assembling protein cage 0626 F068 BG505.SOSIP.T332N_BG505 Circ. permut., Circ. permut., ZM233_NtermH1_4_Ferritin SOSIP,V1V2 swap, T332N, 664, anoparticle  V1V2swap, anoparticle 0627 F069BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626Ker2018_NtermH1_4_Ferritin 0628 F070 BG505.SOSIP.T332N_ BG505Same as Seq_0626 201C, 433C Same as Seq_0626 ZM233_201C433C_NtermH1_4Ferritin 0629 F071 BG505.SOSIP.T332N_ BG505 Same as Seq_0626 201C, 433CSame as Seq_0626 Ker2018_201C433C_NtermH1 _4_Ferritin 0630 F072BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626ZM233 NtermHl 6 Ferritin 0631 F073 BG505.SOSIP.T332N_ BG505Same as Seq_0626 Same as Seq_0626 KER2018 NtermHl 6 Ferritin 0632 F074BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626ZM233_NtermH1_6_longer_Fe rritin 0633 F075 BG505.SOSIP.T332N_ BG505Same as Seq_0626 Same as Seq_0626 KER2018_NtermH1_6_longer_ Ferritin0634 F076 BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626ZM233 NtermHl 12 Ferritin 0635 F077 BG505.SOSIP.T332N_ BG505Same as Seq_0626 Same as Seq_0626 KER2018_NtermH1_12_Ferritin 0636 F078BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626ZM233 NtermHl Tri Ferritin 0637 F079 BG505.SOSIP.T332N_ BG505Same as Seq_0626 Same as Seq_0626 KER2018_NtermH1_Tri_ Ferritin 0638F080 BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626KER2018 Circ LS 0639 F081 BG505.SOSIP.T332N_ BG505 Same as Seq_0626Same as Seq_0626 KER2018 Circ short LS 0640 F082 BG505.SOSIP.T332N_BG505 Same as Seq_0626 Same as Seq_0626 KER2018 Circ long LS 0641 F083BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626 ZM233 Circ LS0642 F084 BG505.SOSIP.T332N_ BG505 Same as Seq_0626 201C, 433CSame as Seq_0626 ZM233 201C433C Circ LS 0643 F085 BG505.SOSIP.T332N_BG505 Same as Seq_0626 Same as Seq_0626 ZM233 NtermHl Tri LS 0644 F086BG505.SOSIP.T332N_ BG505 Same as Seq_0626 Same as Seq_0626KER2018 NtermHl Tri LS 0645 F087 BG505.SOSIP.T332N_ BG505Same as Seq_0626 201C, 433C Same as Seq_0626 ZM233_NtermH1_Tri_201C433C LS 0646 A204 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I225C/V245C DST332N_ T332N I225C/V245C 0647 A205 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, V36C_T606C DS T332N_ T332N V36C/T606C 0648 A206BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F37C_T606C DS T332N_ T332NT37C/T606C 0649 A207 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,V36C_V496C DS T332N_ T332N V36C/V496C 0650 A208 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V36C_P498C DS T332N_ T332N V36C/P498C 0651 A209BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F37C_A497C DS T332N_ T332NT37C/A497C 0652 A210 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,V38C_V496C DS T332N_ T332N V38C/V496C 0653 A211 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, A200C/Q432C DS T332N_ T332N A200C/Q432C 0654 A212BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F202C_M434C DS T332N_ T332NT202C/M434C 0655 A213 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,T202C_M434C_G431F DS, CavF at gp120 C4 T332N_ T332N substitued withT202C/M434C/G431F F and stablize gp120, interprotomer 0656 A214BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F202C_A433C DS T332N_ T332NT202C_A433C 0657 A215 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V182Dsalt bridge at gp120 V2 T332N_ T332N to stablize V2 V182Dby subtitution of D, 0658 A216 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,I251F/L260F hydrophobic core betwwen gp120 T332N_ T332NV2/V3 with double F I251F/L26OF substitution to stabilize V1/V2/V3 0659A217 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I225C/V488C DS T332N_T332N I225C/V488C 0660 A218 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,N478F CavF at gp120 C terminus T332N_ T332N substitued N478Fwith F and stablize gp120 C terminus 0661 A219 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, F163D_Q170R salt bridge at gp120 T332N_ T332NV2 to stablize T163D_Q170R V1/V2 with D and R substitution 0662 A220BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F163C_Q170C DS T332N_ T332NT163C_Q170C 0663 A221 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,309R_Q315D salt bridge at gp120 T332N_ T332N V3 to stablize I309R_Q315DV1/V2/V3 with D and R subtitution 0664 A222 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, 294C_V333C DS T332N_ T332N I294C_V333C 0665 A223BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 294D_V333Rsalt bridge at gp120 T332N_ T332N V3 to stablize I294D_V333RV1/V2/V3 with D and R subtitution 0666 A224 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, G380F_P437F hydrophobic core at gp120 C T332N_ T332Nterminus with double-F G380F_P437F substitutionto stabilize gp120 C terminus 0667 A225 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, G380F CavF at gp120 C4 with F T332N_ T332N substitutionG380F to stablize gp120 0668 A226 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, 3437F CavF at gp120 C terminus with F T332N_ T332Nsubstitution and P437F stablize gp120 C terminus 0669 A227BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V254F_L260F_L261F_hydrophobic core at gp120 C2 with T332N_ T332N G263Ffour-F substitution to V254F_L260F_L261F_G stabilize gp120 263F 0670A228 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V254FCavF at gp120 C2 with F substitution T332N_ T332N to stablize gp120V254F 0671 A229 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L260FCavF at gp120 C2 with F substitution T332NL260F T332N to stablize gp1200672 A230 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L261FCavF at gp120 C2 with F substitution T332N_ T332Nto stablize gp120, interprotomer L261F 0673 A231 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, G263W CavF at gp120 C2 with W substitution T332N_T332N to stablize gp120, interprotomer G263W 0674 A232BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A55F_V75Fhydrophobic core at gp120 C1 with T332N_ T332Ndouble-F substitution to stabilize A55F_V75F gp120 N terminus 0675 A233BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, T77F_V245Fhydrophobic core at gp120 C1/C2 T332N_ T332N with double-F substitutionT77F_V245F to stabilize gp120 N terminus 0676 A234 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, T77F CavF at gp120 C1 with T332N_ T332NF substitution T77F to stablize gp120 N terminus 0677 A235BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S56H_P76E salt bridge at gp120T332N_ T332N C1 to stablize S56H_P76E gp120 N terminus with H and Esubtitution 0678 A236 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A55FCavF at gp120 C1 with T332N_ T332N F substitution A55Fto stablize gp120 N terminus 0679 A237 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A55F_P81W hydrophobic core at gp120 C1 with F T332N_T332N and W substitution to A55F_P81W stabilize gp120 V terminus 0680A238 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L52F_I215Whydrophobic core at gp120 C1/C2 T332N_ T332Nwith double-F substitution to L52F_I215W stabilize gp120 N terminus 0681A239 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 109KO428Esalt bridge at gp120 C1/C4 T332N_ T332N to stablize I109K_Q428Egp120 N/C terminus with K and E substitution 0682 A240BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F257C S375C DS T332N_ T332NT257C S375C 0683 A241 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,A55C T77C DS T332N_ T332N A55C T77C 0684 A242 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L125F_L193W hydrophobic core at gp120 V1/V2 T332N_ T332Nwith F or W substitution to stabilize L125F_L193W gp120Vl/V2 0685 A243BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L125FCavF at gp120 V1/V2. Substitute F to T332N_ T332N stabilize V1/V2/V3L125F 0686 A244 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, N136WCavF at gp120 V1 Substitute W to T332N_ T332N stabilize V1/V2/V3 N136W0687 A245 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, N136W_L154Whydrophobic core at gp120 V1 with T332N_ T332N double W substitutionN136W_L154W to stabilize gp120 Vl/V2 0688 A246 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L193F_N195C_I423C DS, CavF at gp120 V1 substituted withT332N_ T332N F and stabilize V1/V2, interprotomer L193F_N195C_I423C 0689A247 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I323W_I326Fhydrophobic core at gp120 V3 with W T332N_ T332N or F substitution toI323W_I326F stabilize gp120 V1/V2/V3 0690 A248 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, I323W CavF at gp120 V3. Substitute F to T332N_ T332Nstabilize V1/V2/V3 I323W 0691 A249 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, I326F CavF at gp120 V3. Substitute F to T332N_ T332Nstabilize V1/V2/V3 I326F 0692 A250 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, M475F-N478F hydrophobic core at gp120 V3 with W T332N_T332N or F substitution to M475F_N478F stabilize gp120 V1/V2/V3 0693A251 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q130Hsalt bridge at gp120 T332N_ T332N C1 to stabilize Q130Hgp120 N/C terminus with H substitution 0694 A252 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, Q103D_T106K salt bridge at gp120 C1 T332N_ T332Nto stabilize Q103D_T106K gp120 N/C terminus with K and D substitution0695 A253 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, S110H_Q114Esalt bridge at gp120 T332N_ T332N C1 to stabilize $110H_Q114Egp120 N/C terminus with H and E substitution 0696 A254BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, M150F_I326Whydrophobic core at gp120 V1/V3 T332N_ T332N with W or F substitutionM150F_I326W to stabilize gp120 V1/V2/V3 0697 A255 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, L111W CavF at gp120Cl. Substitute W to T332N_T332N stabilize gp120 terminus L111W 0698 A256 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A204F_V208W hydrophobic core at gp120 C2 with W T332N_T332N or F substitution to stabilize gp120 A204F_V208W V1/V2/V3,interprotomer 0699 A257 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,L537C_G41C DS T332N_ T332N L537C_G41C 0700 A258 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V245F CavF at gp120C2. T332N_ T332NSubstitute F to V245F stabilize gp120 V1/V2/V3, interprotomer 0701 A259BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L125W, I195W Cavity filling ofT332N_ T332N  V1V2-V3 interface L125W.I195W near 126-196 disulfide bond0702 A260 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F139W, D140l, G324l,hydrophobic patch between V1V2 T332N_ T332N D325W and V3 near base of V3V2V3_Hyd2 and Variable loop 1 in V1V2 0703 A261 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, Y173W CavF at gp120 V1/V2/V3. 173: T332N_ T332NSubstitute W or F Y173W 0704 A262 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L179W Stabilized V1V2 cap T332N_ T332N L179W 0705 A263BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L175FCavF: V1V2-V3 interface,  T332N_ T332N Substitute F or W, L175F 0706A264 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L175WCavF: V1V2-V3 interface,  T332N_ T332N Substitute F or W, L175W 0707A265 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, E153FCavF: V1V2-V3/gp120core interface, T332N_ T332N Substitute F or W, E153F0708 A266 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, E153WCavF: V1V2-V3/gp120core interface, T332N_ T332N Substitute F or W, E153W0709 A267 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, L154FCavF at gp120Vl/V2. Substitute F, Y T332N_ T332Nor W, stabilize V1/V2/V3 L154F 0710 A268 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L154W CavF at gp120 V1/V2. Substitute F, Y T332N_ T332Nor W, stabilize V1/V2/V3 L154W 0711 A269 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, E164F CavF: inter-protomer, Substitute F or T332N_ T332NW, E164F 0712 A270 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, E164WCavF: inter-protomer, Substitute F or T332N_ T332N W, E164W 0713 A271BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F198FCavF: V1V2-gp120core interface T332N_ T332N T198F 0714 A272BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F202FCavF: V1V2-gp120core interface, T332N_ T332N Substitute F or W, T202F0715 A273 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F202WCavF: V1V2-gp120core interface T332N_ T332N T202W 0716 A274BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A204FCavF: V1V2-gp120core interface, T332N_ T332N Substitute F or W, A204F0717 A275 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I423FCavF: gp120core- V1V2 interface, T332N_ T332N Substitute F or W, I423F0718 A276 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 423WCavF: gp120core- V1V2 interface T332N_ T332N I423W 0719 A277BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, Q432FCavF: stabilize unliganded T332N_ T332N conformation of bridging sheetQ432F region. Substitute F or W, 0720 A278 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, Q432W, Substitute CavF: stabilize unliganded T332N_T332N For W, conformation of bridging Q432W sheet region 0721 A279BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A436MCavF: stabilize unliganded T332N_ T332N conformation of A436Mbridging sheet region, Substitute F, M or W, 0722 A280BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A436F Same as Seq_0721 T332N_T332N A436F 0723 A281 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, A436WSame as Seq_0721 T332N_ T332N A436W 0724 A282 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, A204W CavF: V1V2-gp120core interface, T332N_ T332NSubstitute F or W, A204W 0725 A283 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, N302F CavF: V3- V1V2/gp120core interface, T332N_ T332NSubstitute F or W, N302F 0726 A284 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, M302W CavF: V3- V1V2/gp120core interface, T332N_ T332NSubstitute F or W, N302W 0727 A285 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, 307W V1V2/V3 stabilization/packing, T332N_ T332NSubstitute F or W, I307W 0728 A286 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, I307F V1V2/V3 stabilization/packing/non- T332N_ T332Nbridging sheet, Substitute F or W, I307F 0729 A287 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, F210A α-1 helix/destablize CD4-bound T332N_ T332Nconformation F210A 0730 A288 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,F176W/I323Y CavF at gp120 V1/V2/V3. T332N_ T332N 176: Substitute Y or W;F176W_I323Y 323: F, Y, or W; stabilize V1/V2/V3 0731 A289BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F176W/L154WCavF at gp120 V1/V2. 176: Substitute T332N_ T332N V or W; F176W_L154W154: F, Y, or W; stabilize V1/V2/V3 0732 A290 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, F159Y/L154W CavF at gp120 V1/V2. 159: Substitute T332N_T332N Y or W; F159Y_L154W 154: F, Y, or W; stabilize V1/V2/V3 0733 A291BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F176WCavF at gp120 V1/V2. Substitute Y or T332N_ T332N W, stabilize V1/V2/V3F176W 0734 A292 BG505.R6.664_G41C_L537C BG505 R6, 664 G41C_L537CDS between gp120 and gp41 0735 A293 BG505.R6.664_G41C_A541C BG505R6, 664 G41C_A541C dS between gp120 and gp41 0736 A294BG505.R6.664_P43C_A526C BG505 R6, 664 343C_A526CDS between gp120 and gp41 0737 A295 BG505.R6.664_A73C_G572C BG505R6, 664 A73C_G572C DS between gp120 and gp41 0738 A296BG505.R6.664_I84C_G521C BG505 R6, 664 I84C_G521CDS between gp120 and gp41 0739 A297 BG505.R6.664_V89C_G527C BG505R6, 664 V89C_G527C DS between gp120 and gp41 0740 A298 BG505.IP.R6.664.BG505 IP, R6, 664, A73C_G572C DS between gp120 and gp41 T332N_ T332NA73C_G572C 0741 A299 BG505.IP.R6.664. BG505 IP, R6, 664, I84C_G521CDS between gp120 and gp41 T332N_ T332N I84C_G521C 0742 A300BG505.IP.R6.664. BG505 IP, R6, 664, V89C_G527C DS between gp120 and gp41T332N_ T332N V89C_G527C 0743 A301 BG505.SOSIP.R6.664. BG505 IP, R6, 664,R304C/Q440C DS T332N_ T332N R304C/Q440C 0744 F088 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, I201C/A433C polymeric Fc-fusion protein T332N_T332N I201C/A433C_ polymer hFc fusion 0745 0 ZM233.6 ZM233.6 0746 0Q23.17 Q23.17 0747 0 A244 A244 0748 0 WITO.33 WITO.33 0749 0 ZM53.12ZM53.12 0750 0 CNE58 CNE58 0751 0 3301_V1_C24 3301_V1_C24 0752 Z032foldon domain 0753 Z033 foldon domain 0754 Z034 foldon domain 0755 Z035foldon domain 0756 Z036 Encapsulin subunit 0757 Z037 DNA encodingSEQ ID NO: 352 0758 Z038 BG505 transmembrane domain 0759 Z039DNA encoding BG505 transmembrane domain 0760 Z040Influenza A Hemagglutinin transmembrane domain 0761 Z041DNA encoding Influenza A Hemagglutinin transmembrane domain 0762 Z042Influenza A Neuraminidase transmembrane domain 0763 Z043DNA encoding Influenz a A Neuraminidase transmembrane domain 0764 H034Cap256-SU_bg505- CAP256-SU/BG505 SOSIP, R6, 664 CAP256-SU gp120Heterologous NCgp120 + gp41. chimera With gp120 with SOSIP_(gp41 + gp120-NC (gp41 + ds201-433 from BG505.SOSIP) gp120-NC(Res. 31-45; 478-507) from 3G505. SOSIP 0765 H035 3301_bg505-3301_V1_C24/BG505 SOSIP, R6, 664 3301_V1_C24 gp120 Same as Seq_0764NCgp120 + gp41. chimera with (gp41 + gp120- SOSIP_ MCfrom BG505.SOSIP)ds201-433 0766 H036 ZM53_bg505- ZM53/BG505 SOSIP, R6, 664ZM53 gp120 with Same as Seq_0764 NCgp120 + gp41. chimera(gp41 + gp120-NC from SOSIP_ BG505.SOSIP) ds201-433 0767 H037Cap256-SU_bg505- CAP256-SU/BG505 SOSIP, R6, 664 CAP256-SU gp120BG505 Platform + Int. NCgp120 + gp41. chimera with (gp41 + gp120-NCRes. Set A SOSIP + int_ ds201- 433 from BG505.SOSIP) 0768 H0383301_bg505- 3301_V1_C24/BG505 SOSIP, R6, 664 3301_V1_C24 gp120Same as Seq_0767 NCgp120 + gp41. chimera with (gp41 + gp120-SOSIP + int_ NCfrom BG505.SOSIP) ds201-433 0769 H039 ZM53_bg505-ZM53/BG505 SOSIP, R6, 664 ZM53 gp120 with Same as Seq_ 0767NCgp120 + gp41. chimera (gp41 + gp120-NC from SOSIP + int_ 3G505.SOSIP)ds201-433 0770 H040 CNE58-SUstrandC_bg505- CNE58/BG505 SOSIP, R6, 664CNE58 gp120 with BG505 Platform and NCgp120 + chimera (gp41 + gp120-NCRes. 166-173 gp41. from BG505.SOSIP) from CAP256-SU SOSIP_and (strand C from ds201-433 CAP256-SU) 0771 H041 CNE58-SUstrandC_bg505-CNE58/BG505 SOSIP, R6, 664 CNE58 gp120 with BG505 PlatformNCgp120 + gp41. chimera (gp41 + gp120-NC and Res. 166-173 SOSIP_from BG505.SOSIP) from ds304-440 and (strand C from CAP256-SU CAP256-SU)0772 H042 BG505.SOSIP.664. BG505/CAP45 SOSIP, R6, 664, CAP45 asBG505.SOSIP.664. R6.T332N chimera T332N gp41 sequence R6.T332N_construct with gp41 from CAP45 0773 A302 JR-FLgp140.6R. JR-FLSOSIP, R6, 664, 201C/A433C SOSIP.664. E168K E168K_I201C/A433C 0774 A303JR-FLgp140.6R. JR-FL SOSIP, R6, 664, A433P SOSIP.664. E168K E168K_A433P0775 A304 JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, Q432PE168K_Q432P E168K 0776 A305 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, S174C/A319C E168K_S174C/A319C E168K 0777 A306JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, N195C/A433CE168K_N195C/A433C E168K 0778 A307 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, S199C/A433C E168K_S199C/A433C E168K 0779 A308JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, R304C/Q440CE168K_R304C/Q440C E168K 0780 A309 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, F223W E168K_F223W E168K 0781 A310JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, G473Y E168K_G473Y E168K0782 A311 JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, G431PE168K_G431P E168K 0783 A312 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, N425C_A433C E168K_N425C_A433C E168K 0784 A313JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, V120C_Q315CE168K_V120C_Q315C E168K 0785 A314 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, O203C_L122C E168K_Q203C_L122C E168K 0786 A315 IR- JR-FLSOSIP, R6, 664, 201C/A433C/R304C/ FLgp140.6R.SOSIP.664. E168K Q440CE168K_I201C/A433C/R304C/ Q440C 0787 A316 JR-FLgp140.6R.SOSIP.664. JR-FLSOSIP, R6, 664, 3304C/R440C Lock V3 to gp120 to preventE168K_R304C/R440C E168K exposure/opening 0788 A317JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, Q203C/F317CSame as Seq_0787 E168K_Q203C/F317C E168K 0789 A318JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, L122C/F317CSame as Seq_0787 E168K_L122C/F317C E168K 0790 A319JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, 437C/Y318CSame as Seq_0787 E168K_P437C/Y318C E168K 0791 A320JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, E172C/I307CLocking V3 to V1V2 to prevent E168K_E172C/I307C E168K exposure/opening0792 A321 JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, P206C/Y318CSame as Seq_0787 E168K_P206C/Y318C E168K 0793 A322JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, A174C/T319CSame as Seq_0791 E168K_A174C/T319C E168K 0794 A323JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, S164C/H3O8CSame as Seq_0791 E168K_S164C/H308C E168K 0795 A324JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, F320C/L175CSame as Seq_0791 E168K_T320C/L175C E168K 0796 A325JR-FLgp140.6R.SOSIP.664. JR-FL SOSIP, R6, 664, T320C/P438CSame as Seq_0791 E168K_T320C/P438C E168K 0797 F089KER2008.12_ V1V2V3CAP_ KER2008 V1V2V3 V1V2V3 constructed fer_15ln_gycnanoparticle; at distances circ. permut., defined by the inter-4TVP structure subunit disulfide, ferritin 0798 F090KER2008.12_V1V2V3CAP_ KER2008 Same as Seq_0797 Same as Seq_0797fer_101_n_gyc 0799 F091 KER2008.12_V1V2V3CAP_ KER2008 Same as Seq_0797Same as Seq_0797 fer_101_n_gyc 0800 F092 Q23.17_V1V2V3CAP_ Q23.17Same as Seq_0797 Same as Seq_0797 fer_15ln_gyc 0801 F093Q23.17_V1V2V3CAP_ Q23.17 Same as Seq_0797 Same as Seq_0797 fer_10ln_gyc0802 F094 Q23.17_V1V2V3CAP_ Q23.17 Same as Seq_0797 Same as Seq_0797fer_5ln_gyc 0803 F095 KER2008.12_ KER2008 V1V2V3 Same as Seq_0797 V1V2V3CAP_ Nanoparticle LS_15ln_gyc circ. permut., inter-subunit disulfide, lumazine synthase 0804 F096 KER2008.12_ KER2008Same as Seq_0803 Same as Seq_0797 V1V2V3CAP_LS_10ln_gyc 0805 F097KER2008.12_V1V2V3CAP_ KER2008 Same as Seq_0803 Same as Seq_0797LS_10ln_gyc 0806 F098 Q23.17_V1V2V3CAP_LS_ Q23.17 Same as Seq_0803Same as Seq_0797 15ln_gyc 0807 F099 Q23.17_V1V2V3CAP_LS_ Q23.17Same as Seq_0803 Same as Seq_0797 10ln_gyc 0808 F100Q23.17_V1V2V3CAP_LS_ Q23.17 Same as Seq_0803 Same as Seq_0797 5ln_gyc0809 F101 KER2008.12_ds175_320_ KER2008 V1V2V3 Same as Seq_0797 V1V2V3CAP_ nanoparticle fer_15ln__gyc circ. permut., inter-subunit disulfide, intra-subunit disulfide, Ferritin 0810 F102KER2008.12_ds175_320_ KER2008 Same as Seq_0809 Same as Seq_0797V1V2V3CAP_fer_10ln_gyc 0811 F103 KER2008.12_ds175_320_ KER2008Same as Seq_0809 Same as Seq_0797 V1V2V3CAP_fer_10ln_gyc 0812 F104Q23.17_ds174_319_ Q23.17 Same as Seq_0809 Same as Seq_0797V1V2V3CAP_fer_15ln_gyc 0813 F105 Q23.17_ds174_319_ Q23.17Same as Seq_0809 Same as Seq_0797 V1V2V3CAP_fer_10ln_gyc 0814 F106Q23.17_ds174_319_ Q23.17 Same as Seq_0809 Same as Seq_0797V1V2V3CAP_fer_5ln_gyc 0815 F107 KER2008.12_ds175_ KER2008V1V2V3 nanoparticle Same as Seq_0797 320_ V1V2V3CAP_ LS_15ln_gyccirc. permut., inter- subunit disulfide, lumazine synthase,ntra-subunit disulfide 0816 F108 KER2008.12_ds175_320_ KER2008Same as Seq_0815 Same as Seq_0797 V1V2V3CAP_LS_10ln_gyc 0817 F109KER2008.12_ds175_320_ KER2008 Same as Seq_0815 Same as Seq_0797V1V2V3CAP_LS_10ln_gyc 0818 F110 Q23.17_ds174_319_ Q23.17Same as Seq_0815 Same as Seq_0797 V1V2V3CAP_LS_15ln_gyc 0819 F111Q23.17_ds174_319_ Q23.17 Same as Seq_0815 Same as Seq_0797V1V2V3CAP_LS_10ln_gyc 0820 F112 Q23.17_ds174_319_ Q23.17Same as Seq_0815 Same as Seq_0797 V1V2V3CAP_LS_5ln_gyc 0821 F113BG505 119-1364- BG505 V1V2V3 nanoparticle Same as Seq_0797ln4-296-33H-ln + circ. permut. 151-2054-1ln + ferr 0822 F114CNE58_SU-strandC_119-136 + CNE58 V1V2V3 nanoparticle Same as Seq_0797ln + 296-331 + circ. permut. ln + 151- 205 + 1ln + ferr 0823 F1153301_V1_C24_119-1364-ln + 3301 V1V2V3 nanoparticle Same as Seq_0797296-3314-ln4-151- circ. permut. 205 + 1ln + ferr 0824 F116ZM53-R166W_119-136 + ZM53 V1V2V3 nanoparticle Same as Seq_0797ln + 296-331 + circ. permut. ln + 151- 205 + 1ln + ferr 0825 F117ZM233.6_119-1364-ln4-296- ZM233 V1V2V3 nanoparticle Same as Seq_07973314-ln + 151-2054-1ln4-ferr circ. permut. 0826 F118 BG505_119-136 +BG505 V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151-205 + 5ln + ferr 0827 F119 CNE58_SU-strandC_119-136 + CNE58V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 5ln + ferr 0828 F120 3301_V1_C24_119-136 + 3301V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 5ln + ferr 0829 F121 ZM53-R166W_119-136 + ZM53V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 5ln + ferr 0830 F122 ZM233.6_119-136 + ZM233V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151-205 + 5ln + ferr 0831 F123 BG505_119-136 + BG505V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151-205 + 10ln + ferr 0832 F124 CNE58_SU-strandC_119-136 + CNE58V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 101_n + ferr 0833 F125 3301_V1_C24_119-136 + 3301V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 101_n + ferr 0834 F126 ZM53-R166W_119-136 + ZM53V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 101_n + ferr 0835 F127 ZM233.6_119-136 + ZM233V1V2V3 nanoparticle Same as Seq_0797 ln + 296-331 + circ. permut.ln + 151- 205 + 101_n + ferr 0836 E001 KER2008.12_ V1V2V3CAP_ KER2008V1V2V3 scaffold Same as Seq_0797 lVH8cp_10ln_gyc circ. permut., inter-subunit disulfide, 1VH8 0837 E002 KER2008.12_ V1V2V3CAP_ KER2008V1V2V3 scaffold circ. Same as Seq_0797 lVH8cp_15ln_gyc permut., inter-subunit disulfide, 1VH8 0838 E003 Q23.17_ V1V2V3CAP_ Q23.17V1V2V3 scaffold Same as Seq_0797 lVH8cp_10ln_gyc circ. permut., inter-subunit disulfide, 1VH8 0839 E004 Q23.17_ V1V2V3CAP_ Q23.17V1V2V3 scaffold Same as Seq_0797 lVH8cp_15ln_gyc circ. permut., inter-subunit disulfide, 1VH8 0840 E005 KER2008.12_ds175_ KER2008V1V2V3 scaffold Same as Seq_0797 320_ V1V2V3CAP_ circ. permut., inter-lVH8cp_10ln gyc subunit disulfide, 1VH8, intra-subunit disulfide 0841E006 KER2008.12_ds175_ KER2008 V1V2V3 scaffold Same as Seq_0797320_ V1V2V3CAP_ circ. permut., inter- lVH8cp_15ln subunit disulfide,_gyc 1VH8, intra-subunit disulfide 0842 E007 Q23.17_ds174_ Q23.17V1V2V3 scaffold Same as Seq_0797 319_ V1V2V3CAP_ circ. permut., inter-lVH8cp_10ln_gyc subunit disulfide, 1VH8, intra-subunit disulfide 0843E008 Q23.17_ds174_319_ V1V2V3CAP_ Q23.17 V1V2V3 scaffoldSame as Seq_0797 lVH8cp_15ln_gyc circ. permut., inter-subunit disulfide, 1VH8, intra-subunit disulfide 0844 G004BG505_119-136 + BG505 V1V2V3 trimerization- Same as Seq_0797ln + 296-331 + domain circ. ln + 151- permut. 205 + 3ln + foldon 0845G005 CNE58_SU-strandC_119-136 + CNE58 V1V2V3 trimerizationSame as Seq_0797 ln + 296-331 + -domain circ. ln + 151- permut.205 + 3ln + foldon 0846 G006 3301_V1_C24_119-136 + 3301V1V2V3 trimerization Same as Seq_0797 ln + 296-331 + n + 151--domain circ. 205 + 3ln + foldon permut. 0847 G007 ZM53-R166W_119-136 +ZM53 V1V2V3 trimerization- Same as Seq_0797 ln + 296-331 + domain circ.ln + 151- permut. 205 + 3ln + foldon 0848 G008 ZM233.6_119-136 + ZM233V1V2V3 trimerization- Same as Seq_0797 ln + 296-331 + domain circ.ln + 151- permut. 205 + 3ln + foldon 0849 G009 BG505_119-136 + BG505V1V2V3 trimerization- Same as Seq_0797 ln + 296-331 + domain circ.ln + 151- permut. 205 + 7ln + foldon 0850 G010CNE58_SU-strandC_119-136 + CNE58 V1V2V3 trimerization- Same as Seq_0797ln + 296-331 + domain circ. ln + 151- permut. 205 + 7ln + foldon 0851G011 3301_V1_C24_119-1364-ln + 3301 V1V2V3 trimerization-Same as Seq_0797 296-3314-ln4-151- domain circ. 205 + 7ln + foldonpermut. 0852 G012 ZM53-R166W_119-136 + ZM53 V1V2V3 trimerization-Same as Seq_0797 ln + 296-331 + domain circ. ln + 151- permut.205 + 7ln + foldon 0853 G013 ZM233.6_119-1364-ln + ZM233V1V2V3 trimerization- Same as Seq_0797 296-3314-ln4-151- domain circ.205 + 7ln + foldon permut. 0854 Z Linker 0855 Z 1VH8 Scaffold 1VH8 0856H043 *286.36-chim_ 286.36/BG505 SOSIP, R6, 664, Res. 31-45, 478-507,Same as Seq_0379 d7324.201C-433C chimera 201C/433C 512-664 fromBG505 (“BG505 Platform”), remainder = 286.36 0857 H044 288.38-chim_288.38/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 288.38 0858 H0453988.25-chim_ 3988.25/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = 3988.250859 H046 5768.04-chim_ 5768.04/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder from 5768.04 0860 H047 6101.1-chim_ 5101.1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 6101.1 0861 H048 6535.3-chim_ 5535.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 6535.3 0862 H049 7165.18-chim_ 7165.18/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 7165.18 0863 H050 0013095-2.11-chim_D013095-2.11/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 0013095- 2.11 0864 H051001428-2.42-chim_ D01428-2.42/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera remainder = 001428- 201C/433C2.42 0865 H052 0077_Vl.C16-chim_ D077_Vl.C16/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = D077_V1.C16 0866 H053 00836-2.5-chim_ 00836-2.5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 00836-2.5 0867 H054 0260.v5.c36-chim_D260.v5.c36/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = D260.v5.c36 0868 H0SS0330.v4.c3-chim_ 0330.v4.c3/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 0330.v4.c3 0869 H056 0439.v5.c1-chim_ 3439.v5.c1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 0439.v5.c1 0870 H057 0815.V3.C3-chim_3815.V3.C3/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 0815.V3.C3 0871 H058*0921.V2.C14-chim_ D921.V2.C14/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =0921.V2.C14 0872 H059 *16055-2.3-chim_ 16055-2.3/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 16055-2.3 0873 H060 16845-2.22-chim_ 16845-2.22/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 16845-2.22 0874 H061 16936-2.21-chim_16936-2.21/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 16936-2.21 0875 H062231965.c1-chim_ 231965.C1/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = 231965x10876 H063 235-47-chim_ 235-47/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = 235-470877 H064 242-14-chim_ 242-14/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = 242-140878 H065 247-23-chim_ 247-23/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = 247-230879 H066 25710-2.43-chim_ 25710-2.43/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 25710-2.43 0880 H067 25711-2.4-chim_ 25711-2.4/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 25711-2.4 0881 H068 *25925-2.22-chim_25925-2.22/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 25925-2.22 0882 H06926191-2.48-chim_ 26191-2.48/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 26191-2.48 0883 H070 263-8-chim_ 263-8/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 263-8 0884 H071 269-12-chim_ 269-12/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 269-12 0885 H072 271-11-chim_ 271-11/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 271-11 0886 H073 3016.v5.c45-chim_ 3016.v5.c45/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 3016.v5.c45 0887 H074 3168.V4.C10-chim_3168.V4.C10/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 3168.V4.C10 0888 H075*3301.V1.C24-chim_ 3301.V1.C24/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =3301.V1.C24 0889 H076 3326.V4.C3-chim_ 3326.V4.C3/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 3326.V4.C3 0890 H077 3337.V2.C6-chim_ 3337.V2.C6/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 3337.V2.C6 0891 H078 3365.v2.c20-chim_3365.v2.c20/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 3365.v2.c20 0892 H0793415.V1.c1-chim_ 3415.v1.c1/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 3415.v1.c1 0893 H080 3468.V1.C12-chim_ 3468.V1.C12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 3468.V1.C12 0894 H081 3589.V1.C4-chim_3589.V1.C4/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 3589.V1.C4 0895 H0823637.V5.C3-chim_ 3637.V5.C3/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 3637.V5.C3 0896 H083 3718.v3.c11.chim_ 3718.v3.c11/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 3718.v3.c1l 0897 H084 3817.v2.c59-chim_3817.v2.c59/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 3817.v2.c59 0898 H0853873.V1.C24-chim_ 3873.V1.C24/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =3873.V1.C24 0899 H086 398-Fl_F6_20-chim_ 398-Fl_F6_20/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 398- :1 F6 20 0900 H087 57128.vrcl5-chim_57128.vrcl5/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 57128.vrcl5 0901 H0886095.V1.C10-chim_ 6095.V1.C10/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =6095.V1.C10 0902 4089 *620345.c1-chim_ 520345.C1/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 620345x1 0903 H090 6322.V4.C1-chim_ 5322.V4.C1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 6322.V4.C1 0904 H091 6405.v4.c34-chim_6405.v4.c34/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 201C/433C 6405.v4.c34 0905H092 6471.V1.C16-chim_ 5471.V1.C16/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =6471.V1.C16 0906 H093 6540.V4.c1-chim_ 5540.v4.c1/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 6540.v4.c1 0907 H094 6545.V3.C13-chim_ 6545.V3.C13/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 6545.V3.C13 0908 H095 6545.V4.C1-chim_5545.V4.C1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 6545.V4.C1 0909 H0966631.V3.C10-chim_ 6631.V3.C10/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =6631.V3.C10 0910 H097 6644.V2.C33-chim_ 6644.V2.C33/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 6644.V2.C33 0911 H098 6785.V5.C14-chim_6785.V5.C14/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = 6785.V5.C14 0912 H0996838.V1.C35-chim_ 6838.V1.C35/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =6838.V1.C35 0913 H100 89.6.DG-chim_ 39.6.DG/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 89.6.DG 0914 H101 928-28-chim_ 928-28/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = 928-28 0915 H102 96ZM651.02-chim_ 96ZM651.02/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 96ZM651.02 0916 H103 A03349M1.vrc4a-chim_A03349M1.vrc4a/ SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C BG505 201C/433C remainder = chimera A03349M1.vrc4a 0917H104 *AC10.29-chim_ AC10.29/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = AC10.290918 H105 ADA.DG-chim_ ADA.DG/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = ADA.DG0919 H106 Bal.01-chim_ Bal.01/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Bal.010920 H107 BaL.26-chim_ BaL.26/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = BaL260921 H108 BB201.B42-chim_ BB201.B42/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = BB201.B42 0922 H109 BB539.2B13-chim_ BB539.2B13/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = 3B539.2B13 0923 H110 BG1168.01-chim_BG1168.01/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = BG1168.01 0924 H111*BI369.9A-chim_ BI369.9A/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = BI369.9A0925 H112 BL01.DG-chim_ BL01.DG/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = BL01.DG0926 H113 BR025.9-chim_ BR025.9/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = BR025.90927 H114 BR07.DG-chim_ BR07.DG/BG5O5 chimera SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C 201C/433Cremainder = BR07.DG 0928 H115 BS208.Bl-chim_ BS208.B1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = BS208.B1 0929 H116 BX08.16-chim_ BX08.16/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = BX08.16 0930 H117 *C1080.c3-chim_ C1080.c3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = C1080.c3 0931 H118 C2101.c1-chim_ C2101.C1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = C2101.C1 0932 H119 C3347.c11.chim_ C3347.C11/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_.0379 d7324.201C-433Cchimera 201C/433C remainder = C3347.c1l 0933 H120 *C4118.09-chim_C4118.09/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = C4118.09 0934 H121CAAN.A2-chim_ CAAN.A2/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = CAAN.A20935 H122 CAP210.E8-chim_ CAP210.E8/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = CAP210.E8 0936 H123 CAP244.D3-chim_ CAP244.D3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CAP244.D3 0937 H124 *CAP45.G3-chim_ CAP45.G3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CAP45.G3 0938 H125 *CH038.12-chim_ CH038.12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CH038.12 0939 H126 CH070.1-chim_ CH070.1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CH070.1 0940 H127 *CH117.4-chim_ CH117.4/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CH117.4 0941 H128 CH181.12-chim_ CH181.12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CH181.12 0942 H129 CNElO-chim_ CNE10/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE10 0943 H130 CNE12-chim_ CNE12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE12 0944 H131 CNE14-chim_ CNE14/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE14 0945 H132 CNE15-chim_ CNE15/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE15 0946 H133 CNE3-chim_ CNE3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE3 0947 H134 CNE30-chim_ CNE30/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE30 0948 H135 CNE31-chim_ CNE31/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE31 0949 H136 CNE4-chim_ CNE4/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE4 0950 H137 CNE40-chim_ CNE40/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE40 0951 H138 CNE5-chim_ CNE5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE5 0952 H139 CNE53-chim_ CNE53/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE53 0953 H140 *CNE55-chim_ CNE55/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE55 0954 H141 CNE56-chim_ CNE56/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE56 0955 H142 CNE57-chim_ CNE57/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE57 0956 H143 CNE58-chim_ CNE58/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE58 0957 H144 CNE59-chim_ CNE59/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE59 0958 H145 CNE7-chim_ CNE7/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = CNE7 0959 H146 DJ263.8-chim_ J263.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DJ263.8 0960 H147 DU123.06-chim_ U123.06/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DU123.06 0961 H148 DU151.02-chim_ U151.02/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DU151.02 0962 H149 *DU156.12-chim_ U156.12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DU156.12 0963 H150 DU172.17-chim_ U172.17/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DU172.17 0964 H151 *DU422.01-chim_ U422.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = DU422.01 0965 H152 HO86.8-chim_ HO86.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = HO86.8 0966 H153 HT593.1-chim_ HT593.1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = HT593.1 0967 H154 JRCSF.JB-chim_ JRCSF.JB/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = JRCSF.JB 0968 H155 JRFL.JB-chim_ JRFL.JB/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = JRFL.JB 0969 H156 KER2008.12-chim_KER2008.12/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C chimera 201C/433C remainder = KER2008.12 0970 H157KER2018.11-chim_ KER2018.11/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder =KER2018.11 0971 H158 KNH1209.18-chim_ KNH1209.18/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = KNH1209.18 0972 H159 M02138-chim_ M02138/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MO2138 0973 H160 *MB201.A1-chim_ MB201.A1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MB201.A1 0974 H161 MB539.2B7-chim_ MB539.287/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MB539.2B7 0975 H162 MI369.A5-chim_ M1369.A5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MI369.A5 0976 H163 MN.3-chim_ MN.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MN.3 0977 H164 MS208.A1-chim_ MS208.A1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MS208.A1 0978 H165 *MW965.26-chim_ MW965.26/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = MW965.26 0979 H166 NKU3006.ec1-chim_VKU3006.ec1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C 201C/433C remainder = chimera VKU3006.ec1 0980 H167PVO.04-chim_ VO.04/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = PVO.040981 H168 Q168.a2-chim_ Q168.2/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q168.a20982 H169 Q23.17-chim_ Q23.17/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q23.170983 H170 Q259.17-chim_ Q259.17/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q259.170984 H171 Q461.e2-chim_ 0461.e2/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q461.e20985 H172 Q769.d22-chim_ Q769.d22/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q769.d220986 H173 Q769.h5-chim_ Q769.h5/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q769.h50987 H174 Q842.dl2-chim_ Q842.d12/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = Q842.dl20988 H175 QH0515.01-chim_ QH0515.01/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = QH0515.01 0989 H176 QH0692.42-chim_ QH0692.42/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = QH0692.42 0990 H177 *QH209.14M.A2-chim_QH209.14M.A2/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_0379d7324.201C-433C 201C/433C remainder = chimera QH209.14M.A2 0991 H178R1166.c1-chim_ R1166.C1/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = R1166.C10992 H179 R2184.c4-chim_ R2184.c4/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = R2184.c40993 H180 R3265.c6-chim_ R3265.c6/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = R3265.c60994 H181 REJO.67-chim_ REJO.67/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = REJO.670995 H182 RHPA.7-chim_ HPA.7/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = RHPA.70996 H183 RW020.2-chim_ RW020.2/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = RW020.20997 H184 SC422.8-chim_ SC422.8/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = SC422.80998 H185 SF162.LS-chim_ SF162.LS/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = SF162.LS0999 H186 SO18.18-chim_ SO18.18/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_0379 d7324.201C-433C chimera 201C/433C remainder = SO18.181000 H187 SS1196.01-chim_ SS1196.01/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera 201C/433Cremainder = SS1196.01 1001 H188 T250-4-chim_ T250-4/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T250-4 1002 H189 T251-18-chim_ T251-18/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T251-18 1003 H190 T253-ll-chim_ T253-ll/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T253-11 1004 H191 T255-34-chim_ T255-34/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T255-34 1005 H192 T257-31-chim_ T257-31/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T257-31 1006 H193 T266-60-chim_ T266-60/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T266-60 1007 H194 T278-50-chim_ T278-50/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T278-50 1008 H195 T280-5-chim_ T280-5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T280-5 1009 H196 T33-7-chim_ T33-7/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = T33-7 1010 H197 *TH966.8-chim_ TH966.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TH966.8 1011 H198 TH976.17-chim_ TH976.17/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TH976.17 1012 H199 THRO.18-chim_ THRO.18/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = THRO.18 1013 H200 TRJO.58-chim_ TRJO.58/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TRJO.58 1014 H201 TRO-11-chim_ TRO.11/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TRO.11 1015 H202 TV1.29-chim_ TV1.29/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TV1.29 1016 H203 TZA125.17-chim_ FZA125.17/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TZA125.17 1017 H204 TZBD.02-chim_ FZBD.02/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = TZBD.02 1018 H205 UG021.16-chim_ UG021.16/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = UG021.16 1019 H206 UG024.2-chim_ UG024.2/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = UG024.2 1020 H207 UG037.8-chim_ UG037.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = UG037.8 1021 H208 WITO.33-chim_ WITO.33/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = WITO.33 1022 H209 X2088.c9-chim_ X2088.c9/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = X2088.c9 1023 H210 YU2.DG-chim_ YU2.DG/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = YU2.DG 1024 H211 ZA012.29-chim_ ZA012.29/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZA012.29 1025 H212 *ZM106.9-chim_ ZM106.9/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM106.9 1026 H213 ZM109.4-chim_ ZM109.4/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM109.4 1027 H214 ZM135.10a-chim_ ZM135.10a/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM135.10a 1028 H215 ZM176.66-chim_ ZM176.66/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM176.66 1029 H216 ZM197.7-chim_ ZM197.7/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM197.7 1030 H217 ZM214.15-chim_ ZM214.15/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM214.15 1031 H218 ZM215.8-chim_ ZM215.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM215.8 1032 H219 ZM233.6-chim_ ZM233.6/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM233.6 1033 H220 ZM249.1-chim_ ZM249.1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM249.1 1034 H221 *ZM53.12-chim_ ZM53.12/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM53.12 1035 H222 *ZM55.28a-chim_ ZM55.28a/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_0379 d7324.201C-433C chimera201C/433C remainder = ZM55.28a 1036 H223 6101.1-chim + int_6101.1 + int/BG505 SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579d7324.201C-433C chimera 201C/433C 478-507, 512-664 and Int. Res. set A;remainder-6101.1 1037 H224 Bal.01-chim + int_ Bal.01 + int/BG505SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433Cchimera 201C/433C 478-507, 512-664 and Int. Res. set A; remainder-Bal.011038 H225 BG1168.01-chim + int_ BG1168.01 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder- BG1168.01 1039 H226CAAN.A2-chim + int_ CAAN.A2 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-CAAN.A2 1040 H227DU156.12-chim + int_ U156.12 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-DU156.12 1041 H228DU422.01-chim + int_ DU422.01 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-DU422.01 1042 H229JRCSF.JB-chim + int_ JRCSF.JB + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; SOSIP, R6, 664, remainder-JRCSF.JB1043 H230 JRFL.JB-chim + int_ JRFL.JB + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-JRFL.JB 1044 H231KER2018.11-chim + int_ KER2018.11 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder- KER2018.11 1045 H232PVO.04-chim + int_ PVO.04 + int/BG505 SOSIP, R6, 664, BG505: Res. 31-45,Same as Seq_0579 d7324.201C-433C chimera 201C/433C 478-507, 512-664and Int. Res. set A; remainder-PVO.04 1046 H233 Q168.a2-chim + int_Q168.a2 + int/BG505 SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579d7324.201C-433C chimera 201C/433C 478-507, 512-664 and Int. Res. set A;remainder-Q168. a2 1047 H234 Q23.17-chim + int_ Q23.17 + int/BG505SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433Cchimera 201C/433C 478-507, 512-664 and Int. Res. set A; remainder-Q23.171048 H235 Q769.h5-chim + int_ Q769.h5 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-Q769.h5 1049 H236RW020.2-chim + int_ RW020.2 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-RW020.2 1050 H237THRO.18-chim + int_ THRO.18 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-THRO.18 1051 H238TRJO.58-chim + int_ TRJO.58 + int/BG505 SOSIP, R6, 664,BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433C chimera 201C/433C478-507, 512-664 and Int. Res. set A; remainder-TRJ0.58 1052 H239TRO.11-chim + int_ TRO.11 + int/BG505 SOSIP, R6, 664, BG505: Res. 31-45,Same as Seq_0579 d7324.201C-433C chimera 201C/433C 478-507, 512-664and Int. Res. set A; remainder-TRO.11 1053 H240 YU2.DG-chim + int_YU2.DG + int/BG505 SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579d7324.201C-433C chimera 201C/433C 478-507, 512-664 and Int. Res. set A;remainder-YU2.DG 1054 H241 ZA012.29-chim + int_ ZAO12.29 + int/BG5O5SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433Cchimera 201C/433C 478-507, 512-664 and Int. Res. set A;remainder-ZA012.29 1055 H242 ZM106.9-chim + int_ ZM106.9 + int/BG505SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433Cchimera 201C/433C 478-507, 512-664 and Int. Res. set A;remainder-ZM106.9 1056 H243 ZM55.28a-chim + int_ ZM55.28a + int/BG505SOSIP, R6, 664, BG505: Res. 31-45, Same as Seq_0579 d7324.201C-433Cchimera 201C/433C 478-507, 512-664 and Int. Res. set A; remainder-ZM55.28a 1057 A326 *6101.1.sosip_ 6101.1 SOSIP, R6, 664, 201C, 433Cd7324.201C-433C 1058 A327 *Bal.01.sosip_ Bal.01 SOSIP, R6, 664,201C, 433C d7324.201C-433C 1059 A328 *BG1168.01.sosip_ BG1168.01SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1060 A329 *CAAN.A2.sosip_CAAN.A2 SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1061 A330*DU156.12.sosip_ U156.12 SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1062A331 *DU422.01.sosip_ U422.01 SOSIP, R6, 664, 201C, 433C d7324.201C-433C1063 A332 JRCSF.JB.sosip_ JRCSF.JB SOSIP, R6, 664, 201C, 433Cd7324.201C-433C 1064 A333 *JRFL.JB.sosip_ JRFL.JB SOSIP, R6, 664,201C, 433C d7324.201C-433C 1065 A334 *KER2018.11.sosip_ KER2018.11SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1066 A335 *PVO.04.sosip_PVO.04 SOSIP, R6, 664, 201C, 433C5 d7324.201C-433C 1067 A336*Q168.a2.sosip_ Q168.a2 SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1068A337 *Q23.17.sosip_ Q23.17 SOSIP, R6, 664, 201C, 433C d7324.201C-433C1069 A338 *Q769.h5.sosip_ Q769.h5 SOSIP, R6, 664, 201C, 433C5d7324.201C-433C 1070 A339 *RW020.2.sosip_ 3W020.2 SOSIP, R6, 664,201C, 433C d7324.201C-433C 1071 A340 *THRO.18.sosip_ THRO.18SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1072 A341 *TRJO.58.sosip_TRJO.58 SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1073 A342*TRO.11.sosip_ TRO-11 SOSIP, R6, 664, 201C, 433C d7324.201C-433C 1074A343 *YU2.DG.sosip_ YU2.DG SOSIP, R6, 664, 201C, 433C d7324.201C-433C1075 A344 *ZA012.29.sosip_ ZA012.29 SOSIP, R6, 664, 201C, 433Cd7324.201C-433C 1076 A345 *ZM106.9.sosip_ ZM106.9 SOSIP, R6, 664,201C, 433C d7324.201C-433C 1077 A346 *ZM55.28a.sosip_ ZM55.28aSOSIP, R6, 664, 201C, 433C d7324.201C-433C 1078 H244 6101.1-chim-sc_5101.1/BG505 SOSIP, R6, 664, Seq_0534 linker Chimeric singled7324.201C-433C chimera 201C/433C between 508-511, chain Env with BG505BG505 Platform, gp41ecto/gp120- remainder = ZM55.28a NC “platform” andheterologous gp120 1079 H245 Bal.01-chim-sc_ Bal.01/BG505SOSIP, R6, 664, Seq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = Bal.01 1080 H246BG1168.01-chim-sc_ BG1168.01/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = BG1168.01 1081 H247 CAAN.A2-chim-sc_CAAN.A2/BG505 SOSIP, R6, 664, Seq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, Res.31-45, 478-507, 512-664 from BG505, remainder = CAAN.A2 1082 H248DU156.12-chim-sc_ DU156.12/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = DU156.12 1083 H249 DU422.01-chim-sc_DU422.01/BG505 SOSIP, R6, 664, Seq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, BG505 Platform,remainder = DU422.01 1084 H250 JRCSF.JB-chim-sc_ JRCSF.JB/BG505SOSIP, R6, 664, Seq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = JRCSF.JB 1085H251 JRFL.JB-chim-sc_ JRFL.JB/BG505 SOSIP, R6, 664,5eq_0534 linker between Same as Seq_1078 d7324.201C-433C chimera201C/433C 508-511, Res. 31-45, 478-507, 512-664 from BG505,remainder = JRFL.JB 1086 H252 KER2018.11-chim-sc_ KER2018.11/BG505SOSIP, R6, 664, Seq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = KER2018.11 1087H253 PVO.04-chim-sc_ VO.04/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = PVO.04 1088 H254 Q168.a2-chim-sc_Q168.a2/BG505 SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, BG505 Platform,remainder = Q168.a2 1089 H255 Q23.17-chim-sc_ Q23.17/BG505SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = Q23.17 1090 H256Q769.h5-chim-sc_ Q769.h5/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = Q769.h5 1091 H257 RW020.2-chim-sc_3W020.2/BG505 SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, BG505 Platform,remainder = RW020.2 1092 H258 THRO.18-chim-sc_ THRO.18/BG505SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = THRO.18 1093 H259TRJO.58-chim-sc_ TRJO.58/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = TRJO.58 1094 H260 TRO.11-chim-sc_TRO.11/BG505 SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, BG505 Platform,remainder = TRO.11 1095 H261 YU2.DG-chim-sc_ YU2.DG/BG505SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = YU2.DG 1096 H262ZA012.29-chim-sc_ ZA012.29/BG505 SOSIP, R6, 664, 5eq_0534 linkerSame as Seq_1078 d7324.201C-433C chimera 201C/433C between 508-511,BG505 Platform, remainder = ZA012.29 1097 H263 ZM106.9-chim-sc_ZM106.9/BG505 SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078d7324.201C-433C chimera 201C/433C between 508-511, BG505 Platform,remainder = ZM106.9 1098 H264 ZM55.28a-chim-sc_ ZM55.28a/BG505SOSIP, R6, 664, 5eq_0534 linker Same as Seq_1078 d7324.201C-433C chimera201C/433C between 508-511, BG505 Platform, remainder = ZM55.28a 1099H265 ZM53_bg505- ZM53/BG505 SOSIP, R6, 664, BG505 Platform,Ferritin particle NCgp120 + gp41. chimera 201C/433C; remainder = ZM53,with Chimeric gp140 SOSIP_ 332N ferritin linked with BG505 ds201-to gp41-C gp41ecto/gp120-NC 433_3bve-5ln “platform” andheterologous gp120 1100 H266 CNE55-glyc332_bg505- CNE55/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 chimera 201C/433Cremainder = CNE55, NCgp120 + gp41. ferritin linked to SOSIP_ gp41-Cds201-433_ferr-5ln 1101 H267 P0402_Cll_bg505- 30402_C11/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera201C/433C remainder = SOSIP_ P0402 C11, ds201- ferritin linked433_ferr-5ln to gp41-C 1102 H268 X1193_C1_bg505- X1193_C1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera201C/433C remainder = X1193_C1, SOSIP_ ferritin linked to ds201- gp41-C433_ferr-5ln 1103 H269 DU156.12_bg505- DU156.12/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera 201C/433Cremainder = DU156.12, SOSIP_ ferritin linked to ds201- gp41-C433_ferr-5ln 1104 H270 DU422.01_bg505- DU422.01/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera 201C/433Cremainder = DU422.01, SOSIP_ ferritin linked to ds201- gp41-C433_ferr-5ln 1105 H271 25925-2.22_bg505- 25925-2.22/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera201C/433C remainder = 25925- SOSIP_ 2.22, ferritin ds201- 433_3bve-5lnlinked to gp41-C 1106 H272 3301_V1_C24_bg505- 3301_V1_C24/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera201C/433C remainder = SOSIP_ 33O1_V1_C24, ferritin ds201-linked to gp41-C 433_3bve-5ln 1107 H273 Cap256-SU_bg505- Cap256-SU/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 NCgp120 + gp41. chimera201C/433C remainder = Cap256-SU, SOSIP_ ferritin linked to ds201- gp41-C433_3bve-5ln 1108 H274 CH117.4_332N_ CH117.4_332N/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1099 bg505- chimera 201C/433C remainder =NCgp120 + gp41. CH117.4_332N, ferritin SOSIP_ linked to gp41-Cds201-433_3bve-5ln 1109 H275 CNE58_SU-strandC_ CNE58_SU-strand C/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 bg505- chimera201C/433C Res. 166-173 (strand C) NCgp120 + gp41. From CAP256-SU, SOSIP_remainder = CNE58, ds201-433_3bve-5ln Ferritin linked to gp41-C 1110H276 KER2018.11_bg505- KER2018.11/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 NCgp120 + gp41. chimera 201C/433C remainder = SOSIP_KER2018.11, ferritin ds201- linked to gp41-C 433_3bve-5ln 1111 H277ZM233.6_bg505- ZM233.6/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 NCgp120 + int + gp41.SOSIP_ chimera 201C/433Cremainder = ZM233.6, ds201- ferritin linked to 433_3bve-5ln gp41-C 1112H278 ZM53_bg505- ZM53/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 NCgp120 + gp41. chimera 201C/433C remainder = ZM53,SOSIP_ ferritin linked to ds201- gp41-C 433_3bve-5ln 1113 H27945_01dG5_bg505- dG5 from d45/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 NCgp120 + gp41. chimera 201C/433C remainder = dG5,SOSIP_ ferritin linked to ds201- gp41-C 433_3bve_5ln 1114 H280231965.c1-chim_ 231965.C1/BG505 SOSIP, R6, 664, BG505 Platform,Chimeric gp140 with d7324.201C-433C.mi-cl-min chimera 201C/433CRes. Set B (Res. 133; gp41ecto/gp120-NC 134; “platform” and 164;BG505 Res. Set B, 169; and heterologous 308; gpl20 316) from BG505(“BG505 Res. Set B”), remainder = 231965x1 1115 H281 288.38-chim_288.38/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = 288.38 1116 H282 3415.v1.c1-chim_ 3415.v1.c1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = 3415.v1.c1 1117 H283 3817.v2.c59-chim_ 3817.v2.c59/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = 3817.v2.c59 1118 H284 57128.vrcl5-chim_ 57128.vrcl5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = 57128.vrcl5 1119 H285 6535.3-chim_ 5535.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = 6535.3 1120 H286 89.6.DG-chim_ 89.6.DG/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, remainder = 89.6.DG 1121 H287A03349M1.vrc4a-chim_ A03349M1.vrc4a/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min 201C/433CBG505 Res. Set B, chimera remainder = A03349M1. vrc4a 1122 H288Bal.01-chim_ Bal.01/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera 201C/433CBG505 Res. Set B, remainder = Bal.01 1123 H289 BaL.26-chim_ BaL.26/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = BaL.26 1124 H290 BG1168.01-chim_ BG1168.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = BG1168.01 1125 H291 BR07.DG-chim_ BR07.DG/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = BR07.DG 1126 H292 CNE10-chim_ CNE10/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, remainder = CNE10 1127 H293 CNE30-chim_CNE30/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = CNE30 1128 H294 CNE4-chim_ CNE4/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, emainder = CNE4 1129 H295 JRCSF.JB-chim_JRCSF.JB/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = JRCSF.JB 1130 H296 JRFLJJB-chim_ JRFL.JB/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = JRFL.JB 1131 H297 MB539.2B7-chim_ MB539.2B7/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = MB539.2B7 1132 H298 MN.3-chim_ MN.3/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, remainder = MN.3 1133 H299 NKU3006.ec1-chim_\KU3006.ec1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min 201C/433C BG505 Res. Set B, chimeraremainder = NKU3006.ec1 1134 H300 PVO.04-chim_ VO.04/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = PVO.04 1135 H301 Q259.17-chim_ Q259.17/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, remainder = 0, 259.17 1136 H302QH0692.42-chim_ QH0692.42/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera 201C/433CBG505 Res. Set B, remainder = QH0692.42 1137 H303 SF162.LS-chim_5F162.LS/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = SF162.LS 1138 H304 SS1196.01-chim_ SS1196.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = SS1196.01 1139 H305 F266-60-chim_ F266-60/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = T266-60 1140 H306 F280-5-chim_ F280-5/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1114 d7324.201C-433C.mi-cl-min chimera201C/433C BG505 Res. Set B, remainder = T280-5 1141 H307 UG024.2-chim_UG024.2/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = UG024.2 1142 H308 ZM215.8-chim_ ZM215.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1114d7324.201C-433C.mi-cl-min chimera 201C/433C BG505 Res. Set B,remainder = ZM215.8 1143 H309 231965.c1-chim_ 231965.C1/BG505SOSIP, R6, 664, BG505 Platform, Chimeric gp140 withd7324.201C-433C.mi-cl1 chimera 201C/433C Res. Set C gp41ecto/(Res. 49; 133; gp120-NC “platform” and 134; 149; 150;Res. Set C from BG505, and 151; 152; 164; heterologous gp120 169; 188;190; 211; 223; 252; 281; 293; 308; 316; 336; 340; 352; 360; 362; 363;369; 372; 393; 410; 432; 442; 444; 446; 474; 476) from BG505(“BG505 Res. Set C”), remainder = ZM215.8 1144 H310 288.38-chim_288.38/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C Res. Set C from BG505,remainder = 288.38 1145 H311 3415.v1.c1-chim_ 3415.v1.c1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = 3415.v1.c1 1146 H3123817.v2.c59-chim_ 3817.v2.c59/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, emainder = 3817.v2.c59 1147 H313 57128.vrcl5-chim_57128.vrcl5/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = 57128.vrcl5 1148 H314 6535.3-chim_ 6535.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = 6535.3 1149 H31589.6.DG-chim_ 89.6.DG/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, remainder = 89.6.DG 1150 H316 A03349M1.vrc4a-chim_A03349M1.vrc4a/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 201C/433C BG505 Res. Set C, chimeraremainder = A03349M1.vrc4a 1151 H317 Bal.01-chim_ Bal.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = Bal.01 1152 H318BaL.26-chim_ BaL.26/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, remainder = BaL.26 1153 H319 BG1168.01-chim_BG1168.01/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = BG1168.01 1154 H320 BR07.DG-chim_ BR07.DG/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = BR07.DG 1155 H321CNE10-chim_ CNE10/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = CNE10 1156 H322 CNE30-chim_ CNE30/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera201C/433C BG505 Res. Set C, remainder = CNE30 1157 H323 CNE4-chim_CNE4/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = CNE4 1158 H324 JRCSF.JB-chim_ JRCSF.JB/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera201C/433C BG505 Res. Set C, remainder = JRCSF.JB 1159 H325 JRFL.JB-chim_JRFL.JB/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = JRFL.JB 1160 H326 MB539.2B7-chim_ MB539.287/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = MB539.2B7 1161 H327MN.3-chim_ MN.3/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = MN.3 1162 H328 NKU3006.ec1-chim_ VKU3006.ec1/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1201C/433C BG505 Res. Set C, chimera remainder = NKU3006.ec1 1163 H329PVO.04-chim_ PVO.04/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, remainder = PVO.04 1164 H330 Q259.17-chim_3259, 17/86505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = Q259.17 1165 H331 QH0692.42-chim_ QH0692.42/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = QH0692.42 1166 H332SF162.LS-chim_ 5F162.LS/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, emainder = SF162.LS 1167 H333 SS1196.01-chim_SS1196.01/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = SS1196.01 1168 H334 T266-60-chim_ T266-60/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = T266-60 1169 H335T280-5-chim_ T280-5/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1143 d7324.201C-433C.mi-cl1 chimera 201C/433CBG505 Res. Set C, remainder = T280-5 1170 H336 UG024.2-chim_UG024.2/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1143d7324.201C-433C.mi-cl1 chimera 201C/433C BG505 Res. Set C,remainder = UG024.2 1171 H337 ZM215.8-chim_ ZM215.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1143 d7324.201C-433C.mi-cl1chimera 201C/433C BG505 Res. Set C, remainder = ZM215.8 1172 H338231965.c1-chim_ 231965.C1/BG505 SOSIP, R6, 664, BG505 Platform,Chimeric gp140 with d7324.201C-433C.mi-cl1-2 chimera 201C/433CRes. Set C, + Res. Set D gp41ecto/gp120-NC (46; 60; 62; 63; 84;“platform” and 85; 87; 99; 102; 130; Res. Sets C and D132; 135; 153; 158; from BG505, and 160; 161; 165; 166;heterologous gp120 167; 171; 172; 173; 175; 177; 178; 181;184; 185; 189; 202; 232; 234; 236; 240; 268; 269; 270; 271;275; 277; 287; 289; 292; 295; 297; 305; 315; 317; 319; 322;328; 330; 332; 333; 334; 335; 337; 339; 343; 344; 345; 346;347; 350; 351; 357; 371; 375; 379; 387; 389; 394; 411; 412;413; 415; 424; 426; 429; 440; 460; 461; 465; 475; 477) fromBG505 (+37 BG505 Res. Set D+38 ), remainder = 231965x1 1173 H339288.38-chim_ 288.38/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = 288.38 1174 H340 3415.v1.c1-chim_3415.v1.c1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = 3415.v1.c1 1175 H341 3817.v2.c59-chim_ 3817.v2.c59/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = 3817.v2.c59 1176 H342 57128.vrcl5-chim_ 57128.vrcl5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = 57128.vrcl5 1177 H343 6535.3-chim_ 5535.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = 6535.3 1178 H344 89.6.DG-chim_ 89.6.DG/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera201C/433C BG505 Res. Sets C + D, remainder = 89.6.DG 1179 H345A03349M1.vrc4a-chim_ A03349M1.vrc4a/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 201C/433CBG505 Res. Sets C + D, chimera remainder = A03349M1.vrc4a 1180 H346Bal.01-chim_ Bal.01/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = Bal.01 1181 H347 BaL.26-chim_BaL.26/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,emainder = BaL.26 1182 H348 BG1168.01-chim_ BG1168.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = BG1168.01 1183 H349 BR07.DG-chim_ BR07.DG/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = BR07.DG 1184 H350 CNE10-chim_ CNE10/BG505 SOSIP, R6, 664,BG505 Platform BG505 Res. Same as Seq_1172 d7324.201C-433C.mi-cl1-2chimera 201C/433C Sets C + D, remainder = CNE10 1185 H351 CNE30-chim_CNE30/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = CNE30 1186 H352 CNE4-chim_ CNE4/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera201C/433C BG505 Res. Sets C + D, remainder = CNE4 1187 H353JRCSF.JB-chim_ JRCSF.JB/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = JRCSF.JB 1188 H354 JRFL.JB-chim_JRFL.JB/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = JRFL.JB 1189 H355 MB539.2B7-chim_ MB539.287/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = MB539.2B7 1190 H356 MN.3-chim_ MN.3/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera201C/433C BG505 Res. Sets C + D, remainder = MN.3 1191 H357NKU3006.ec1-chim_ NKU3006.ec1/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = NKU3006.ec1 1192 H358 PVO.04-chim_VO.04/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = PVO.04 1193 H359 Q259.17-chim_ Q259.17/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera201C/433C BG505 Res. Sets C + D, remainder = Q259.17 1194 H360QH0692.42-chim_ QH0692.42/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = QH0692.42 1195 H361 SF162.LS-chim_SF162.LS/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = SF162.LS 1196 H362 SS1196.01-chim_ SS1196.01/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = SS1196.01 1197 H363 T266-60-chim_ F266-60/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = T266-60 1198 H364 T280-5-chim_ T280-5/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera201C/433C BG505 Res. Sets C + D, remainder = T280-5 1199 H365UG024.2-chim_ UG024.2/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1172 d7324.201C-433C.mi-cl1-2 chimera 201C/433CBG505 Res. Sets C + D, remainder = UG024.2 1200 H366 ZM215.8-chim_ZM215.8/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1172d7324.201C-433C.mi-cl1-2 chimera 201C/433C BG505 Res. Sets C + D,remainder = ZM215.8 1201 H367 0921.V2.C14-chim_ D921.V2.C14/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 201C-433C_5ln-ferrchimera 201C/433C gp140 remainder = 0921.V2.C14, ferritinlinked to gp41-C 1202 H368 16055-2.3-chim_ 16055-2.3/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 201C-433C_5ln-ferrchimera 201C/433C gp140 remainder = 16055-2.3, ferritin linked to gp41-C1203 H369 286.36-chim_ 286.36/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =286.36, ferritin linked to gp41-C 1204 H370 620345.c1-chim_620345.C1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =620345.c1, ferritin linked to gp41-C 1205 H371 6545.V4.C1-chim_6545.V4.C1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainders6545.V4.Cl, ferritin linked to gp41-C 1206 H372 AC10.29-chim_AC10.29/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder = AC10.29, ferritinlinked to gp41-C 1207 H373 BI369.9A-chim_ BI369.9A/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433Cgp140 remainder = 31369.9A, ferritin linked to gp41-C 1208 H374C1080.c3-chim_ C1080.c3/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =C1080.c3, ferritin linked togp41-C 1209 H375 C4118.09-chim_C4118.09/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =C4118.09, ferritin linked to gp41-C 1210 H376 CAP45.G3-chim_CAP45.G3/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =CAP45.G3, ferritinlinked to gp41-C 1211 H377 CH038.12-chim_ CH038.12/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433Cgp140 remainder = CH038.12, ferritin linked to gp41-C 1212 H378CH117.4-chim_ CH117.4/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =CH117.4, ferritin linked to gp41-C 1213 H379 MB201.A1-chim_MB201.A1/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =MB201.A1, ferritin linked togp41-C 1214 H380 MW965.26-chim_MW965.26/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =MW965.26, ferritin linked to gp41-C 1215 H381 QH209.14M.A2-chim_QH209.14M.A2/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr 201C/433C gp140 remainder = Q.H209.14M.A2, ferritinchimera linked to gp41-C 1216 H382 TH966.8-chim_ TH966.8/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1099 201C-433C_5ln-ferrchimera 201C/433C gp140 remainder = TH966.8, ferritin linked to gp41-C1217 H383 ZM106.9-chim_ ZM106.9/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1099 201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =ZM106.9, ferritin linked to gp41-C 1218 H384 ZM55.28a-chim_ZM55.28a/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1099201C-433C_5ln-ferr chimera 201C/433C gp140 remainder =ZM55.28a, ferritin linked to gp41-C 1219 0 BI369.9A BI369.9A 1220 0MB201.A1 MB201.A1 1221 0 QH209.14M.A2 QH209.14M.A2 1222 0 0921.V2.C141921.V2.C14 1223 0 16055-2.3 16055-2.3 1224 0 25925-2.22 25925-2.22 12250 286.36 286.36 1226 0 CAP45.G3 CAP45.G3 1227 0 CNE58 CNE58 1228 0DU156.12 DU156.12 1229 0 DU422.01 DU422.01 1230 0 MW965.26 MW965.26 12310 ZM53.12 ZM53.12 1232 0 ZM55.28a ZM55.28a 1233 0 ZM106.9 ZM106.9 1234 03301.V1.C24 3301.V1.C24 1235 0 6545.V4.C1 5545.V4.C1 1236 0 620345.c1520345.c1 1237 0 C1080.C3 C1080.C3 1238 0 C4118.09 C4118.09 1239 0 CNE55CNE55 1240 0 TH966.8 rH966.8 1241 0 4C10.29 4C10.29 1242 0 CH038.12CH038.12 1243 3 CH117.4 CH117.4 1244 A347 *CH505, BG505 CH505/BG505SOSIP, R6, 664, I201C/A433C chimera, chimera T332NSOSIP.R6.664 I201C/A433CAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNATASNSSIIEGMKNCSFNITTELRDKREKKNALFYKLDIVQDGNSSQYRLINCNTSVCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITNNVKTIIVHLNESVKIECTRPNNKTRTSIRIGPGQAFYATGQVIGDIREAYCNINESKWNETLQRVSKKLKEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTETNSTRTITIHCRIKQIINMWQEVGRCMYAPPIAGNITCISNITGLLLTRDGGKNNTETFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTV0ARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1245A348 BG505.SOSIP. BG505 SOSIP, R6, 664, V134F/L175M/1322M/1326MCavity Filling/ R6.664. T332N, Hydrophobic core T332N_ I201C/A433CI201C/A433C/V134F/L1 75M/I322M/I326M 1246 A349 BG505.SOSIP. BG505Same as Seq_1245 V134F/1322Y/1326M Cavity Filling/ R6.664.Hydrophobic core T332N_ I201C/A433C/V134F/I3 22Y/I326M 1247 A350BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V1341/1175W/1322F/1326MCavity Filling/ T332NI201C Hydrophobic core /A433C/V134I/L175W/I322F/I326M 1248 A351 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V134F/N136W/M150H/1326M Cavity Filling/ Hydrophobic core T332N_I201C/A433C/V134F/N 136W/M150H/I326M 1249 A352 BG505.SOSIP. BG505Same as Seq_1245 V134F/N136W/M150F/1326L Cavity Filling/ R6.664.Hydrophobic core T332N_ I201C/A433C/V134F/N 136W/M150F/I326L 1250 A353BG505.SOSIP. BG505 Same as Seq_1245 V1341/N136W/M150F/1326LCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/V134I/N136W/M150F/I326L 1251 A354 BG505.SOSIP.R6. BG505 Same as Seq_1245V134F/N136F/M150L/1326M Cavity Filling/ 664. Hydrophobic core T332N_I201C/A433C/V134F/N 136F/M150L/I326M 1252 A355 BG505.SOSIP. BG505Same as Seq_1245 L154M/N300M/N302M/T320L Cavity Filling/ R6.664.Hydrophobic core T332N_ I201C/A433C/L154M/N 300M/N302M/T320L 1253 A356BG505.SOSIP. BG505 Same as Seq_1245 L154F/N300L/N302M/T320LCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/L154F/N300L/N302M/T320L 1254 A357 BG505.SOSIP. BG505 Same as Seq_1245L154W/N300L/N302G/T320F Cavity Filling/ R6.664. Hydrophobic core T332N_I201C/A433C/L154W/N 300L/N302G/T320F 1255 A358 BG505.SOSIP. BG505Same as Seq_1245 V120F/Q203M/Y318M Cavity Filling/ R6.664.Hydrophobic core T332N_ I201C/A433C/V120F/Q 203M/Y318M 1256 A359BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V1201/Q203M/Y318WCavity Filling/ T332NI201C/A433C/ Hydrophobic core V120I/Q2 03M/Y318W1257 A360 BG505.SOSIP. BG505 Same as Seq_1245 V120W/Q203M/Y318WCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/V120W/Q203M/Y318W 1258 A361 BG505.SOSIP. BG505 Same as Seq_1245 V120F/Q315MCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/V120F/Q 315M1259 A362 BG505.SOSIP. BG505 Same as Seq_1245 V120W/Q315FCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/V120W/ Q315F1260 A363 BG505.SOSIP. BG505 Same as Seq_1245 Y177W/1420MCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/Y177W/ I420M1261 A364 BG505.SOSIP. BG505 Same as Seq_1245 Y177W/Q328F/I420MCavity Filling/ R6.664. Hydrophobic core T332N_ I201C/A433C/Y177W/Q328F/I420M 1262 A365 BG505.SOSIP. BG505 Same as Seq_1245L116M/M426F/Q432M Cavity Filling/ R6.664. Hydrophobic core T332N_I201C/A433C/L116M/ M426F/Q432M 1263 A366 BG505.SOSIP.R6.664. BG505Same as Seq_1245 L116M/M426F/Q432W Cavity Filling/ T332NI201C/Hydrophobic core A433C/L116M/ M426F/Q432W 1264 A367 BG505.SOSIP.R6.664.BG505 Same as Seq_1245 VI426F/Q432L Cavity Filling/ T332N_Hydrophobic core I201C/A433C/M426F/Q 432L 1265 A368 BG505.SOSIP.R6.664.BG505 Same as Seq_1245 V134F/L175M/1322M/ Cavity Filling/ T332N_1326M/N136W/M Hydrophobic core I201C/A433C/V134F/ 150H L175M/I322M/I326M/N136W/M150H 1266 A369 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V1341/1175W/1322F/ Cavity Filling/ T332N_ 1326L/N136W/M15Hydrophobic core I201C/A433C/V134I/ 0F L175W/I322F/ I326L/N136W/M150F1267 A370 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V120F/Q203M/Y318M/Q315M Cavity Filling/ T332N_ Hydrophobic coreI201C/A433C/V120F/ Q203M/ Y318M/Q315M 1268 A371 BG505.SOSIP.R6.664.BG505 Same as Seq_1245 V120W/Q203M/Y318W/Q315F Cavity Filling/ T332N_Hydrophobic core I201C/A433C/V120W/ Q203M/Y318W/Q315F 1269 A372BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L154M/N300M/N302M/Cavity Filling/ T332N_ T320L/Y177W/I4 Hydrophobic coreI201C/A433C/L154M/N 20M 300M/N302M/T320L/ Y177W/I420M 1270 A373BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L154W/N300L/N302G/Cavity Filling/ T332N_ T320F/Y177W/Q3 Hydrophobic coreI201C/A433C/L154W/N 28F/I420M 300L/N302G/T320F/ Y177W/Q328F/I420M 1271A374 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 E153F Cavity FillingT332N_ I201C/A433C/E153F 1272 A375 BG505.SOSIP.R6.664. BG505Same as Seq_1245 E153W Cavity Filling T332N_ I201C/A433C/E153W 1273 A376BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L154F Cavity Filling T332N_I201C/A433C/L154F 1274 A377 BG505.SOSIP.R6.664. BG505 Same as Seq_1245L154W Cavity Filling T332N_ I201C/A433C/L154W 1275 A378BG505.SOSIP.R6.664. BG505 Same as Seq_1245 E164F Cavity Filling T332N_I201C/A433C/E164F 1276 A379 BG505.SOSIP.R6.664. BG505 Same as Seq_1245E164W Cavity Filling T332N_ I201C/A433C/E164W 1277 A380BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V172F Cavity Filling T332N_I201C/A433C/V172F 1278 A381 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V172W Cavity Filling T332N_ I201C/A433C/V172W 1279 A382BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L175F Cavity Filling T332N_I201C/A433C/L175F 1280 A383 BG505.SOSIP.R6.664. BG505 Same as Seq_1245F176W Cavity Filling T332N_ I201C/A433C/F176W 1281 A384BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L179F Cavity Filling T332N_I201C/A433C/L179F 1282 A385 BG505.SOSIP.R6.664. BG505 Same as Seq_1245L179W Cavity Filling T332N_ I201C/A433C/L179W 1283 A386BG505.SOSIP.R6.664. BG505 Same as Seq_1245 Y191F Cavity Filling T332N_I201C/A433C/Y191F 1284 A387 BG505.SOSIP.R6.664. BG505 Same as Seq_1245Y191W Cavity Filling T332N_ I201C/A433C/Y191W 1285 A388BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L193F Cavity Filling T332N_I201C/A433C/L193F 1286 A389 BG505.SOSIP.R6.664. BG505 Same as Seq_1245L193W Cavity Filling T332N_ I201C/A433C/L193W 1287 A390BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L194W Cavity Filling T332N_I201C/A433C/I194W 1288 A391 BG505.SOSIP.R6.664. BG505 Same as Seq_1245T198F Cavity Filling T332N_ I201C/A433C/T198F 1289 A392BG505.SOSIP.R6.664. BG505 Same as Seq_1245 T198W Cavity Filling T332N_I201C/A433C/T198W 1290 A393 BG505.SOSIP.R6.664. BG505 Same as Seq_1245T202F Cavity Filling T332N_ I201C/A433C/T202F 1291 A394BG505.SOSIP.R6.664. BG505 Same as Seq_1245 T202W Cavity Filling T332N_I201C/A433C/T202W 1292 A395 BG505.SOSIP.R6.664. BG505 Same as Seq_1245A204F Cavity Filling T332N_ I201C/A433C/A204F 1293 A396BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A204W Cavity Filling T332N_1201C/A433C/A204W 1294 A397 BG505.SOSIP.R6.664. BG505 Same as Seq_1245N302F Cavity Filling T332N_ I201C/A433C/N302F 1295 A398BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V302W Cavity Filling T332N_I201C/A433C/N302W 1296 A399 BG505.SOSIP.R6.664. BG505 Same as Seq_1245R304F Cavity Filling T332N_ I201C/A433C/R304F 1297 A400BG505.SOSIP.R6.664. BG505 Same as Seq_1245 R304W Cavity Filling T332N_I201C/A433C/R304W 1298 A401 BG505.SOSIP.R6.664. BG505 Same as Seq_1245307F Cavity Filling T332N_ I201C/A433C/I307F 1299 A402BG505.SOSIP.R6.664. BG505 Same as Seq_1245 307W Cavity Filling T332N_I201C/A433C/I307W 1300 A403 BG505.SOSIP.R6.664. BG505 Same as Seq_1245Q315F Cavity Filling T332N_ I201C/A433C/Q315F 1301 A404BG505.SOSIP.R6.664. BG505 Same as Seq_1245 Q315W Cavity Filling T332N_I201C/A433C/Q315W 1302 A405 BG505.SOSIP.R6.664. BG505 Same as Seq_1245I423F Cavity Filling T332N_ I201C/A433C/I423F 1303 A406BG505.SOSIP.R6.664. BG505 Same as Seq_1245 I430F Cavity Filling T332N_I201C/A433C/I430F 1304 A407 BG505.SOSIP.R6.664. BG505 Same as Seq_1245I430W Cavity Filling T332N_ I201C/A433C/I430W 1305 A408BG505.SOSIP.R6.664. BG505 Same as Seq_1245 Q432F Cavity Filling T332N_I201C/A433C/Q432F 1306 A409 BG505.SOSIP.R6.664. BG505 Same as Seq_1245Q432W Cavity Filling T332N_ I201C/A433C/Q432W 1307 A410BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A436M Cavity Filling T332N_I201C/A433C/A436M 1308 A411 BG505.SOSIP.R6.664. BG505 Same as Seq. 1245A436F Cavity Filling T332N_ I201C/A433C/A436F 1309 A412BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A436W Cavity Filling T332N_I201C/A433C/A436W 1310 A413 BG505.SOSIP. BG505 Same as Seq. 1245L125W/I194W Cavity Filling R6.664. T332NI201C/ A433C/L125W/ I194W 1311A414 BG505.SOSIP. BG505 Same as Seq. 1245 F139W/D140I/ Cavity FillingR6.664. G324I/D325W T332N_ I201C/ A433C/T139W/D 140I/G324I/D325W 1312A415 BG505.SOSIP. BG505 Same as Seq_1245 F210A Destabilization R6.664.of CD4-induced T332N_ conformation I201C/ A433C/F210A 1313 A416BG505.SOSIP. BG505 Same as Seq_1245 F210S Destabilization R6.664.of CD4-induced T332N_ conformation I201C/ A433C/F210S 1314 A417BG505.SOSIP. BG505 Same as Seq_1245 Q432P Destabilization R6.664.of CD4-induced T332N_ conformation I201C/ A433C/Q432P 1315 A418BG505.SOSIP.R6.664. BG505 Same as Seq_1245 T538C/Q652C Disulfide T332N_I201C/A433C/T538C/Q 652C 1316 A419 BG505.SOSIP.R6.664. BG505Same as Seq_1245 R304C/Q440C Disulfide T332NI201C/A433C/R304C/Q 440C1317 A420 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 F159YCavity Filling T332N_ I201C/A433C/F159Y 1318 A421 BG505.SOSIP.R6.664.BG505 Same as Seq_1245 I323Y Cavity Filling T332N_ I201C/A433C/I323Y1319 A422 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 F159Y/I323YCavity Filling T332N_ I201C/A433C/F159Y/I3 23Y 1320 A423BG505.SOSIP.R6.664. BG505 Same as Seq_1245 F223W Cavity Filling T332N_I201C/A433C/F223W 1321 A424 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V580L Cavity Filling T332N_ I201C/A433C/V580L 1322 A425BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V583L Cavity Filling T332N_I201C/A433C/V583L 1323 A426 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V580L/V583L Cavity Filling T332NI201C/A433C/ V580L/V583L 1324 A427BG505.SOSIP.R6.664. BG505 Same as Seq_1245 W69P helix 0 disruptionT332N_ I201C/A433C/W69P 1325 A428 BG505.SOSIP.R6.664. BG505Same as Seq_1245 V68P helix 0 disruption T332N_ I201C/A433C/V68P 1326A429 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 T71P helix 0 disruptionT332N_ I201C/A433C/T71P 1327 A430 BG505.SOSIP.R6.664. BG505Same as Seq_1245 V75W Cavity Filling T332N_ I201C/A433C/V75W 1328 A431BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V75F Cavity Filling T332N_I201C/A433C/V75F 1329 A432 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V75M Cavity Filling T332N_ I201C/A433C/V75M 1330 A433BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V208W Cavity Filling T332N_I201C/A433C/V208W 1331 A434 BG505.SOSIP.R6.664. BG505 Same as Seq_1245V208F Cavity Filling T332N_ I201C/A433C/V208F 1332 A435BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A58C/T77C Disulfide T332N_I201C/A433C/A58C/T7 7C 1333 A436 BG505.SOSIP.R6.664. BG505Same as Seq_1245 D57C/T77C Disulfide T332N_ I201C/A433C/D57C/T7 7C 1334A437 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 N67P helix 0 disruptionT332N_ I201C/A433C/N67P 1335 A438 BG505.SOSIP.R6.664. BG505Same as Seq_1245 466P helix 0 disruption T332N_ I201C/A433C/H66P 1336A439 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 N67P/H66Phelix 0 disruption T332NI201C/A433C/ N67P/H66P 1337 A440BG505.SOSIP.R6.664. BG505 Same as Seq_1245 W112I Cavity Filling T332N_I201C/A433C/W112I 1338 A441 BG505.SOSIP.R6.664. BG505 Same as Seq_1245W112M Cavity Filling T332N_ I201C/A433C/W112M 1339 A442BG505.SOSIP.R6.664. BG505 Same as Seq_1245 W427I Cavity Filling T332N_I201C/A433C/W427I 1340 A443 BG505.SOSIP.R6.664. BG505 Same as Seq_1245W427M Cavity Filling T332N_ I201C/A433C/W427M 1341 A444BG505.SOSIP.R6.664. BG505 Same as Seq_1245 3429NDestabilization of CD4 binding site T332N_ I201C/A433C/R429N 1342 A445BG505.SOSIP.R6.664. BG505 Same as Seq_1245 3429LDestabilization of CD4 binding site T332N_ I201C/A433C/R429L 1343 A446BG505.SOSIP.R6.664. BG505 Same as Seq_1245 R429L/W427MDestabilization of CD4 binding site T332N_ I201C/A433C/R429L/W 427M 1344A447 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 G431GC/S199C DisulfideT332N_ I201C/A433C/G431GC/ S199C 1345 A448 BG505.SOSIP.R6.664. BG505Same as Seq_1245 V120W Cavity Filling T332N_ I201C/A433C/V120W 1346 A449BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A316W Cavity Filling T332N_I201C/A433C/A316W 1347 A450 BG505.SOSIP.R6.664. BG505 Same as Seq_1245I309W Cavity Filling T332N_ I201C/A433C/I309W 1348 A451BG505.SOSIP.R6.664. BG505 Same as Seq_1245 S115W Cavity Filling T332N_I201C/A433C/S115W 1349 A452 BG505.SOSIP.R6.664. BG505 Same as Seq_1245P118W Cavity Filling T332N_ I201C/A433C/P118W 1350 A453BG505.SOSIP.R6.664. BG505 Same as Seq_1245 A70Y Cavity Filling T332N_I201C/A433C/A70Y 1351 A454 BG505.SOSIP.R6.664. BG505 Same as Seq_1245A70F Cavity Filling T332N_ I201C/A433C/A70F 1352 A455BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L111Y Cavity Filling T332N_I201C/A433C/L111Y 1353 A456 BG505.SOSIP.R6.664. BG505 Same as Seq_1245L111F Cavity Filling T332N_ I201C/A433C/L111F 1354 A457BG505.SOSIP.R6.664. BG505 Same as Seq_1245 T202P disrupt bridging sheet/T332N_ destabilizing I201C/A433C/T202P CD4 bound state 1355 A458BG505.SOSIP.R6.664. BG505 Same as Seq_1245 V120T disrupt bridging sheet/T332N_ destabilizing I201C/A433C/V120T CD4 bound state 1356 A459BG505.SOSIP.R6.664. BG505 Same as Seq_1245 I573Tdestabilize gp41 helix bundle T332N_ I201C/A433C/I573T 1357 A460BG505.SOSIP.R6.664. BG505 Same as Seq_1245 G594Ndestabilize gp41 helix bundle T332N_ I201C/A433C/G594N 1358 A461BG505.SOSIP.R6.664. BG505 Same as Seq_1245 I573T/G594Ndestabilize gp41 helix bundle T332NI201C/A433C/I573T/G5 94N 1359 A462BG505.SOSIP.R6.664. BG505 Same as Seq_1245 I573T/G594N/K574Edestabilize gp41 helix bundle T332N_ I201C/A433C/I573T/G5 94N/K574E 1360A463 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 I573T/G594N/K574Tdestabilize gp41 helix bundle T332N_ I201C/A433C/I573T/G5 94N/K574T 1361A464 BG505.SOSIP.R6.664. BG505 Same as Seq_1245 K117W Cavity FillingT332N_ I201C/A433C/K117W 1362 A465 BG505.SOSIP.R6.664. BG505Same as Seq_1245 S110W Cavity Filling T332N_ I201C/A433C/S110W 1363 A466BG505.SOSIP.R6.664. BG505 Same as Seq_1245 L544Y Cavity Filling T332N_I201C/A433C/L544Y 1364 A467 BG505.SOSIP.R6.664. BG505 Same as Seq_1245L544Y/L537Y Cavity Filling T332N_ I201C/A433C/L544Y/ L537Y 1365 A468BG505.SOSIP.R6.664. BG505 Same as Seq_1245 544Y/F223W Cavity FillingT332NI201C/A433C/L544Y/ F223W 1366 A469 BG505.SOSIP.R6.664. BG505Same as Seq_1245 544Y/L537Y/F223W Cavity Filling T332N_I201C/A433C/L544Y/L5 37Y/F223W 1367 A470 BG505.SOSIP.R6.664. BG505Same as Seq_1245 Delta_P206 proline removal. T332N_ Removal of groundI201C/A433C/Delta_ state destabilization/ P206 flexibility 1368 A471BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, I194W/T198M/N425FCavity Filling/ T332N_ T332N Hydrophobic core I194W/T198M/N425F 1369A472 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, F198M/N425FCavity Filling/ T332N_ T332N Hydrophobic core T198M/N425F 1370 A473BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 194W/T198Y/ Cavity Filling/T332N_ T332N N425F Hydrophobic core I194W/T198Y/N425F 1371 A474BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, 194F/T198L Cavity Filling//N425W Hydrophobic core T332N_ T332N I194F/T198L/N425W 1372 A475BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V134F/L175M/ Cavity Filling/T332N_ T332N 1322M/1326M Hydrophobic core V134F/L175M/I322M/I 326M 1373A476 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V134F/1322Y/Cavity Filling/ T332N_ T332N 1326M Hydrophobic core V134F/I322Y/I326M1374 A477 BG505.SOSIP. BG505 SOSIP, V1341/1175W/ Cavity Filling/ R6.664.R6, 664, 1322F/1326M Hydrophobic core T332N_ T332N V134I/ L175W/I322F/I326M 1375 A478 BG505.SOSIP.R6.664. BG505 SOSIP, V134F/N136W/Cavity Filling/ T332N_ R6, 664, M150H/1326M Hydrophobic coreV134F/N136W/M150H T332N /I326M 1376 A479 BG505.SOSIP. BG505SOSIP, R6, 664, V134F/N136W/ Cavity Filling/ R6.664. M150F/1326LHydrophobic core T332N_ T332N V134F/ N136W/M150F/ I326L 1377 A480BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V1341/N136W/ Cavity Filling/T332N_ T332N M150F/1326L Hydrophobic core V134I/ N136W/M150F/ I326L 1378A481 BG505.SOSIP. BG505 SOSIP, V134F/N136F/ Cavity Filling/ R6.664.R6, 664, M150L/1326M Hydrophobic core T332N_ T332N V134F/ N136F/M150L/T326M 1379 A482 BG505.SOSIP.R6.664. BG505 SOSIP, L154M/N300M/N302M/Cavity Filling/ T332N_ R6, 664, T320L Hydrophobic core L154M/ T332NN300M/N302M /T320L 1380 A483 BG505.SOSIP. BG505 SOSIP, L154F/N300L/Cavity Filling/ R6.664. R6, 664, N302M/T320L Hydrophobic core T332N_T332N L154F/ N300L/N302M/ T320L 1381 A484 BG505.SOSIP. BG505 SOSIP, R6,L154W/N300L/ Cavity Filling/ R6.664. 664, N302G/T320F Hydrophobic coreT332N_ T332N L154W/ N300L/N302G/ T320F 1382 A485 BG505.SOSIP. BG505SOSIP, R6, 664, V120F/Q203M/Y318M Cavity Filling/ R6.664. T332NHydrophobic core T332N_ V120F/Q203M/Y318M 1383 A486 BG505.SOSIP.R6.664.BG505 SOSIP, R6, 664, V1201/Q203M/Y318W Cavity Filling/ T332N_ T332NHydrophobic core V120I/Q203M/Y318W 1384 A487 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, V120W/Q203M/Y318W Cavity Filling/ T332N_ T332NHydrophobic core V120W/Q203M/Y318W 1385 A488 BG505.SOSIP.R6.664. BG505SOSIP, V120F/Q315M Cavity Filling/ T332N_ R6, 664, Hydrophobic coreV120F/Q315M T332N 1386 A489 BG505.SOSIP.R6.664. BG505 SOSIP, V120W/Q315FCavity Filling/ T332N_ R6, 664, Hydrophobic core V120W/Q315F T332N 1387A490 BG505.SOSIP.R6.664. BG505 SOSIP, Y177W/I420M Cavity Filling/ T332N_R6, 664, Hydrophobic core Y177W/I420M T332N SOSIP, 1388 A491BG505.SOSIP.R6.664. BG505 SOSIP, Y177W/Q328F/1420M Cavity Filling/T332N_ R6, 664, Hydrophobic core Y177W/Q328F/I420M T332N 1389 A492BG505.SOSIP.R6.664. BG505 SOSIP, L116M/M426F/Q432M Cavity Filling/T332N_ R6, 664, Hydrophobic core L116M/M426F/Q432M T332N 1390 A493BG505.SOSIP.R6.664. BG505 SOSIP, L116M/M426F/Q432W Cavity Filling/T332N_ R6, 664, Hydrophobic core L116M/M426F/Q432W T332N 1391 A494BG505.SOSIP.R6.664. BG505 SOSIP, M426F/Q432L Cavity Filling/ T332N_R6, 664, Hydrophobic core M426F/Q432L T332N 1392 A495BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V134F/L175M/1322M/Cavity Filling/ T332N_ T332N 1326M/N136W/M Hydrophobic core V134F/ L50HL175M/I322M/I 326M/N136W/M150H 1393 A496 BG505.SOSIP. BG505 SOSIP, R6,V134l/L175W/l322F/ Cavity Filling/ R6.664. 664, l326L/N136W/M15Hydrophobic core T332N_ T332N DF V134I/ L175W/I322F/I3 26L/N136W/M150F1394 A497 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664, V120F/Q203M/Cavity Filling/ T332N_ T332N Y318M/Q315M Hydrophobic core V120F/Q203M/Y318M/ Q315M 1395 A498 BG505.SOSIP.R6.664. BG505 SOSIP, R6, 664,V120W/Q203M/ Cavity Filling/ T332N_ T332N Y318W/Q315F Hydrophobic coreV120W/Q203M/Y318 W/Q315F 1396 A499 BG505.SOSIP.R6.664. BG505SOSIP, R6, 664, L154M/N300M/N302M/ Cavity Filling/ T332N_ T332NT320L/Y177W/I4 Hydrophobic core L154M/N300M/N302M 20M /T320L/Y177W/I420M1397 A500 BG505.SOSIP. BG505 SOSIP, R6, 664, L154W/N300L/N302G/Cavity Filling/ R6.664. T332N T320F/Y177W/Q3 Hydrophobic core T332N_28F/I420M L154W/N300L/N302G/ r320F/Y177W/Q, 328F/l420M 1398 A501703010505.TF. 703010505.TF SOSIP, R6, 664 I201C/A433C sosip_d7324, R6, 664, 1201C/A433C 1399 A502 703010505.TF. 703010505.TFSOSIP, R6, 664 I201C/A433C trimer association SOSip_ domain mutationsd7324_ tad, R6, 664, I201C/A433C/E47D/K49E/ V65K/E106T/E429R/R432Q/E500R 1400 A503 286.36.sosip_ 286.36 SOSIP, R6, 664 I201C/A433C d73241401 A504 288.38.sosip_ 288.38 SOSIP, R6, 664 I201C/A433C d7324 1402A505 3988.25.sosip_ 3988.25 SOSIP, R6, 664 I201C/A433C d7324 1403 A5065768.04.sosip_ 5768.04 SOSIP, R6, 664 I201C/A433C d7324 1404 A5076101.1.sosip_ 5101.1 SOSIP, R6, 664 I201C/A433C d7324 1405 A5086535.3.sosip_ 5535.3 SOSIP, R6, 664 I201C/A433C d7324 1406 A5097165.18.sosip_ 7165.18 SOSIP, R6, 664 I201C/A433C d7324 1407 A51000130952.11.sosip_ 130952.11 SOSIP, R6, 664 I201C/A433C d7324 1408 A5110014282.42.sosip_ 14282.42 SOSIP, R6, 664 I201C/A433C d7324 1409 A5120077_V1.C16.sosip_ 1077_V1.C16 SOSIP, R6, 664 I201C/A433C d7324 1410A513 008362.5.sosip_ 3362.5 SOSIP, R6, 664 I201C/A433C d7324 1411 A5140260.v5.c36.sosip_ 1260.v5.c36 SOSIP, R6, 664 I201C/A433C d7324 1412A515 0330.v4.c3.sosip_ 1330.v4.c3 SOSIP, R6, 664 I201C/A433C d7324 1413A516 D439.v5.c1.sosip_ 1439.v5.c1 SOSIP, R6, 664 I201C/A433C d7324 1414A517 0815.V3.C3.sosip_ 1815.V3.C3 SOSIP, R6, 664 I201C/A433C d7324 1415A518 0921.V2.C14.sosip_ 1921.V2.C14 SOSIP, R6, 664 I201C/A433C d73241416 A519 160552.3.sosip_ 160552.3 SOSIP, R6, 664 I201C/A433C d7324 1417A520 168452.22.sosip_ 168452.22 SOSIP, R6, 664 I201C/A433C d7324 1418A521 169362.21.sosip_ 169362.21 SOSIP, R6, 664 I201C/A433C d7324 1419A522 231965.c1.sosip_ 231965.C1 SOSIP, R6, 664 I201C/A433C d7324 1420A523 23547.sosip_ 23547 SOSIP, R6, 664 I201C/A433C d7324 1421 A52424214.sosip_ 24214 SOSIP, R6, 664 I201C/A433C d7324 1422 A52524723.sosip_ 24723 SOSIP, R6, 664 I201C/A433C d7324 1423 A526257102.43.sosip_ 257102.43 SOSIP, R6, 664 I201C/A433C d7324 1424 A527257112.4.sosip_ 257112.4 SOSIP, R6, 664 I201C/A433C d7324 1425 A528259252.22.sosip_ 259252.22 SOSIP, R6, 664 I201C/A433C d7324 1426 A529261912.48.sosip_ 261912.48 SOSIP, R6, 664 I201C/A433C d7324 1427 A5302638.sosip_ 2638 SOSIP, R6, 664 I201C/A433C d7324 1428 A531 26912.sosip_26912 SOSIP, R6, 664 I201C/A433C d7324 1429 A532 27111.sosip_ 27111SOSIP, R6, 664 I201C/A433C d7324 1430 A533 3016.v5.c45.sosip_3016.v5.c45 SOSIP, R6, 664 I201C/A433C d7324 1431 A5343168.V4.C10.sosip_ 3168.V4.C10 SOSIP, R6, 664 I201C/A433C d7324 1432A535 3301.V1.C24.sosip_ 3301.V1.C24 SOSIP, R6, 664 I201C/A433C d73241433 A536 3326.V4.C3.sosip_ 3326.V4.C3 SOSIP, R6, 664 I201C/A433C d73241434 A537 3337.V2.C6.sosip_ 3337.V2.C6 SOSIP, R6, 664 I201C/A433C d73241435 A538 3365.v2.c20.sosip_ 3365.v2.c20 SOSIP, R6, 664 I201C/A433Cd7324 1436 A539 3415.v1.c1.sosip_ 3415.v1.c1 SOSIP, R6, 664 I201C/A433Cd7324 1437 A540 3468.V1.C12.sosip_ 3468.V1.C12 SOSIP, R6, 664I201C/A433C d7324 1438 A541 3589.V1.C4.sosip_ 3589.V1.C4 SOSIP, R6, 664I201C/A433C d7324 1439 A542 3637.V5.C3.sosip_ 3637.V5.C3 SOSIP, R6, 664I201C/A433C d7324 1440 A543 3718.v3.c11.sosip_ 3718.v3.c1lSOSIP, R6, 664 I201C/A433C d7324 1441 A544 3817.v2.c59.sosip_3817.v2.c59 SOSIP, R6, 664 I201C/A433C d7324 1442 A5453873.V1.C24.sosip_ 3873.V1.C24 SOSIP, R6, 664 I201C/A433C d7324 1443A546 398F1_F6_20.sosip_ 398F1_F6_20 SOSIP, R6, 664 I201C/A433C d73241444 A547 57128.vrc15.sosip_ 57128.vrc15 SOSIP, R6, 664 I201C/A433Cd7324 1445 A548 6095.V1.C10.sosip_ 5095.V1.C10 SOSIP, R6, 664I201C/A433C d7324 1446 A549 620345.c1.sosip_ 520345.c1 SOSIP, R6, 664I201C/A433C d7324 1447 A550 6322.V4.Cl.sosip_ 5322.V4.C1 SOSIP, R6, 664I201C/A433C d7324 1448 A551 6405.v4.c34.sosip_ 5405.v4.c34SOSIP, R6, 664 I201C/A433C d7324 1449 A552 6471.V1.C16.sosip_5471.V1.C16 SOSIP, R6, 664 I201C/A433C d7324 1450 A553 6540.v4.c1.sosip_5540.v4.c1 SOSIP, R6, 664 I201C/A433C d7324 1451 A554 6545.V3.C13.sosip_5545.V3.C13 SOSIP, R6, 664 I201C/A433C d7324 1452 A555 6545.V4.C1.sosip_5545.V4.C1 SOSIP, R6, 664 I201C/A433C d7324 1453 A556 6631.V3.C10.sosip_5631.V3.C10 SOSIP, R6, 664 I201C/A433C d7324 1454 A5576644.V2.C33.sosip_ 5644.V2.C33 SOSIP, R6, 664 I201C/A433C d7324 1455A558 6785.V5.C14.sosip_ 5785.V5.C14 SOSIP, R6, 664 I201C/A433C d73241456 A559 6838.V1.C35.sosip_ 5838.V1.C35 SOSIP, R6, 664 I201C/A433Cd7324 1457 A560 89.6.DG.sosip_ 39.6.DG SOSIP, R6, 664 I201C/A433C d73241458 A561 92828.sosip_ 92828 SOSIP, R6, 664 I201C/A433C d7324 1459 A56296ZM651.02.sosip_ 96ZM651.02 SOSIP, R6, 664 I201C/A433C d7324 1460 A563A03349M1.vrc4a.sosip_ A03349M1.vrc4a SOSIP, R6, 664 I201C/A433C d73241461 A564 4C10.29.sosip_ AC10.29 SOSIP, R6, 664 I201C/A433C d7324 1462A565 ADA.DG.SOSip_ ADA.DG SOSIP, R6, 664 I201C/A433C d7324 1463 A566Bal.01.sosip_ Bal.01 SOSIP, R6, 664 I201C/A433C d7324 1464 A567BaL.26.sosip_ BaL.26 SOSIP, R6, 664 I201C/A433C d7324 1465 A568BB201.B42.sosip_ BB201.B42 SOSIP, R6, 664 I201C/A433C d7324 1466 A569BB539.2B13.sosip_ BB539.2B13 SOSIP, R6, 664 I201C/A433C d7324 1467 A570BG1168.01.sosip_ BG1168.01 SOSIP, R6, 664 I201C/A433C d7324 1468 A571BI369.9A.sosip_ BI369.9A SOSIP, R6, 664 I201C/A433C d7324 1469 A572BLOl.DG.sosip_ BL01.DG SOSIP, R6, 664 I201C/A433C d7324 1470 A573BR025.9.SOSip_ BRO25.9 SOSIP, R6, 664 I201C/A433C d7324 1471 A574BRO7.DG.sosip_ BR07.DG SOSIP, R6, 664 I201C/A433C d7324 1472 A575BS208.B1.sosip_ BS208.B1 SOSIP, R6, 664 I201C/A433C d7324 1473 A576BX08.16.sosip_ BX08.16 SOSIP, R6, 664 I201C/A433C d7324 1474 A577C1080.c3.sosip_ C1080.C3 SOSIP, R6, 664 I201C/A433C d7324 1475 A578C2101.c1.sosip_ C2101.c1 SOSIP, R6, 664 I201C/A433C d7324 1476 A579C3347.c11.sosip_ C3347.cH SOSIP, R6, 664 I201C/A433C d7324 1477 A580C4118.09.sosip_ C4118.09 SOSIP, R6, 664 I201C/A433C d7324 1478 A581CAAN.A2.sosip_ CAAN.A2 SOSIP, R6, 664 I201C/A433C d7324 1479 A582CAP210.E8.sosip_ CAP210.E8 SOSIP, R6, 664 I201C/A433C d7324 1480 A583CAP244.D3.SOSip_ CAP244.D3 SOSIP, R6, 664 I201C/A433C d7324 1481 A584CAP45.G3.sosip_ CAP45.G3 SOSIP, R6, 664 I201C/A433C d7324 1482 A585CH038.12.sosip_ CH038.12 SOSIP, R6, 664 I201C/A433C d7324 1483 A586CH070.1.sosip_ CH070.1 SOSIP, R6, 664 I201C/A433C d7324 1484 A587CH117.4.sosip_ CH117.4 SOSIP, R6, 664 I201C/A433C d7324 1485 A588CH181.12.sosip_ CH181.12 SOSIP, R6, 664 I201C/A433C d7324 1486 A589CNE10.sosip_ CNE10 SOSIP, R6, 664 I201C/A433C d7324 1487 A590CNE12.sosip_ CNE12 SOSIP, R6, 664 I201C/A433C d7324 1488 A591CNE14.sosip_ CNE14 SOSIP, R6, 664 I201C/A433C d7324 1489 A592CNE15.sosip_ CNE15 SOSIP, R6, 664 I201C/A433C d7324 1490 A593CNE3.sosip_ CNE3 SOSIP, R6, 664 I201C/A433C d7324 1491 A594 CNE30.sosip_CNE30 SOSIP, R6, 664 I201C/A433C d7324 1492 A595 CNE31.sosip_ CNE31SOSIP, R6, 664 I201C/A433C d7324 1493 A596 CNE4.sosip_ CNE4SOSIP, R6, 664 I201C/A433C d7324 1494 A597 CNE40.sosip_ CNE40SOSIP, R6, 664 I201C/A433C d7324 1495 A598 CNE5.sosip_ CNE5SOSIP, R6, 664 I201C/A433C d7324 1496 A599 CNE53.sosip_ CNE53SOSIP, R6, 664 I201C/A433C d7324 1497 A600 CNE55.sosip_ CNE55SOSIP, R6, 664 I201C/A433C d7324 1498 A601 CNE56.sosip_ CNE56SOSIP, R6, 664 I201C/A433C d7324 1499 A602 CNE57.sosip_ CNE57SOSIP, R6, 664 I201C/A433C d7324 1500 A603 CNE58.sosip_ CNE58SOSIP, R6, 664 I201C/A433C d7324 1501 A604 CNE59.sosip_ CNE59SOSIP, R6, 664 I201C/A433C d7324 1502 A605 CNE7.sosip_ CNE7SOSIP, R6, 664 I201C/A433C d7324 1503 A606 DJ263.8.sosip_ J263.8SOSIP, R6, 664 I201C/A433C d7324 1504 A607 DU123.06.sosip_ U123.06SOSIP, R6, 664 I201C/A433C d7324 1505 A608 DU151.02.sosip_ U151.02SOSIP, R6, 664 I201C/A433C d7324 1506 A609 DU156.12.sosip_ U156.12SOSIP, R6, 664 I201C/A433C d7324 1507 A610 DU172.17.sosip_ U172.17SOSIP, R6, 664 I201C/A433C d7324 1508 A611 DU422.01.sosip_ U422.01SOSIP, R6, 664 I201C/A433C d7324 1509 A612 HO86.8.sosip_ H086.8SOSIP, R6, 664 I201C/A433C d7324 1510 A613 HT593.1.sosip_ HT593.1SOSIP, R6, 664 I201C/A433C d7324 1511 A614 JRCSF.JB.sosip_ JRCSF.JBSOSIP, R6, 664 I201C/A433C d7324 1512 A615 JRFL.JB.sosip_ JRFL.JBSOSIP, R6, 664 I201C/A433C d7324 1513 A616 KER2008.12.sosip_ KER2008.12SOSIP, R6, 664 I201C/A433C d7324 1514 A617 KER2018.11.sosip_ KER2018.11SOSIP, R6, 664 I201C/A433C d7324 1515 A618 KNH1209.18.sosip_ KNH1209.18SOSIP, R6, 664 I201C/A433C d7324 1516 A619 M02138.sosip_ M02138SOSIP, R6, 664 I201C/A433C d7324 1517 A620 MB201.A1.sosip_ MB201.A1SOSIP, R6, 664 I201C/A433C d7324 1518 A621 MB539.2B7.sosip_ MB539.287SOSIP, R6, 664 I201C/A433C d7324 1519 A622 MI369.A5.sosip_ M1369.A5SOSIP, R6, 664 I201C/A433C d7324 1520 A623 MN.3.sosip_ MN.3SOSIP, R6, 664 I201C/A433C d7324 1521 A624 MS208.A1.sosip_ MS208.A1SOSIP, R6, 664 I201C/A433C d7324 1522 A625 MW965.26.sosip_ MW965.26SOSIP, R6, 664 I201C/A433C d7324 1523 A626 NKU3006.ec1.sosip_NKU3006.ec1 SOSIP, R6, 664 I201C/A433C d7324 1524 A627 PVO.04.sosip_PVO.04 SOSIP, R6, 664 I201C/A433C d7324 1525 A628 Q168.a2.sosip_ Q168.a2SOSIP, R6, 664 I201C/A433C d7324 1526 A629 Q23.17.sosip_ Q23.17SOSIP, R6, 664 I201C/A433C d7324 1527 A630 Q259.17.sosip_ Q259.17SOSIP, R6, 664 I201C/A433C d7324 1528 A631 Q461.e2.sosip_ 0461.e2SOSIP, R6, 664 I201C/A433C d7324 1529 A632 Q769.d22.sosip_ Q769.d22SOSIP, R6, 664 I201C/A433C d7324 1530 A633 Q769.h5.sosip_ Q769.h5SOSIP, R6, 664 I201C/A433C d7324 1531 A634 Q842.dl2.sosip_ Q842.d12SOSIP, R6, 664 I201C/A433C d7324 1532 A635 QH0515.01.sosip_ QH0515.01SOSIP, R6, 664 I201C/A433C d7324 1533 A636 QH0692.42.sosip_ QH0692.42SOSIP, R6, 664 I201C/A433C d7324 1534 A637 QH209.14M.A2.sosip_QH209.14M.A2 SOSIP, R6, 664 I201C/A433C d7324 1535 A638 R1166.c1.sosip_R166.c1 SOSIP, R6, 664 I201C/A433C d7324 1536 A639 R2184.c4.sosip_R2184.c4 SOSIP, R6, 664 I201C/A433C d7324 1537 A640 R3265.c6.sosip_33265.c6 SOSIP, R6, 664 I201C/A433C d7324 1538 A641 REJO.67.sosip_REJO.67 SOSIP, R6, 664 I201C/A433C d7324 1539 A642 RHPA.7.sosip_ RHPA.7SOSIP, R6, 664 I201C/A433C d7324 1540 A643 RW020.2.sosip_ RW020.2SOSIP, R6, 664 I201C/A433C d7324 1541 A644 SC422.8.sosip_ dsC422.8SOSIP, R6, 664 I201C/A433C d7324 1542 A645 SF162.LS.SOSip_ SF162.LSSOSIP, R6, 664 I201C/A433C d7324 1543 A646 SO18.18.sosip_ S018.18SOSIP, R6, 664 I201C/A433C d7324 1544 A647 SS1196.01.sosip_ SS1196.01SOSIP, R6, 664 I201C/A433C d7324 1545 A648 F2504.sosip_ F2504SOSIP, R6, 664 I201C/A433C d7324 1546 A649 F25118.sosip_ 25118SOSIP, R6, 664 I201C/A433C d7324 1547 A650 F25311.sosip_ F25311SOSIP, R6, 664 I201C/A433C d7324 1548 A651 F25534.sosip_ F25534SOSIP, R6, 664 I201C/A433C d7324 1549 A652 F25731.sosip_ F25731SOSIP, R6, 664 I201C/A433C d7324 1550 A653 F26660.sosip_ F26660SOSIP, R6, 664 I201C/A433C d7324 1551 A654 F27850.sosip_ F27850SOSIP, R6, 664 I201C/A433C d7324 1552 A655 F2805.sosip_ F2805SOSIP, R6, 664 I201C/A433C d7324 1553 A656 F337.sosip_ F337SOSIP, R6, 664 I201C/A433C d7324 1554 A657 TH966.8.sosip_ TH966.8SOSIP, R6, 664 I201C/A433C d7324 1555 A658 TH976.17.sosip_ TH976.17SOSIP, R6, 664 I201C/A433C d7324 1556 A659 THRO.lS.SOSip_ THRO.18SOSIP, R6, 664 I201C/A433C d7324 1557 A660 TRJO.SS.sosip_ TRJO.58SOSIP, R6, 664 I201C/A433C d7324 1558 A661 TRO.11.sosip_ TRO.11SOSIP, R6, 664 I201C/A433C d7324 1559 A662 rvi.29.sosip_ TV1.29SOSIP, R6, 664 I201C/A433C d7324 1560 A663 FZA125.17.sosip_ TZA125.17SOSIP, R6, 664 I201C/A433C d7324 1561 A664 rZBD.02.sosip_ TZBD.02SOSIP, R6, 664 I201C/A433C d7324 1562 A665 UG021.16.sosip_ JGO21.16SOSIP, R6, 664 I201C/A433C d7324 1563 A666 UG024.2.sosip_ UG024.2SOSIP, R6, 664 I201C/A433C d7324 1564 A667 UG037.8.sosip_ UG037.8SOSIP, R6, 664 I201C/A433C d7324 1565 A668 WITO.33.sosip_ WITO.33SOSIP, R6, 664 I201C/A433C d7324 1566 A669 X2088.c9.sosip_ X2088.c9SOSIP, R6, 664 I201C/A433C d7324 1567 A670 YU2.DG.sosip_ YU2.DGSOSIP, R6, 664 I201C/A433C d7324 1568 A671 ZA012.29.sosip_ ZA012.29SOSIP, R6, 664 I201C/A433C d7324 1569 A672 ZM106.9.sosip_ ZM106.9SOSIP, R6, 664 I201C/A433C d7324 1570 A673 ZM109.4.sosip_ ZM109.4SOSIP, R6, 664 I201C/A433C d7324 1571 A674 ZM135.10a.sosip_ ZM135.10aSOSIP, R6, 664 I201C/A433C d7324 1572 A675 ZM176.66.sosip_ ZM176.66SOSIP, R6, 664 I201C/A433C d7324 1573 A676 ZM197.7.sosip_ ZM197.7SOSIP, R6, 664 I201C/A433C d7324 1574 A677 ZM214.15.sosip_ ZM214.15SOSIP, R6, 664 I201C/A433C d7324 1575 A678 ZM215.8.sosip_ ZM215.8SOSIP, R6, 664 I201C/A433C d7324 1576 A679 ZM233.6.sosip_ ZM233.6SOSIP, R6, 664 I201C/A433C d7324 1577 A680 ZM249.1.sosip_ ZM249.1SOSIP, R6, 664 I201C/A433C d7324 1578 A681 ZM53.12.sosip_ ZM53.12SOSIP, R6, 664 I201C/A433C d7324 1579 A682 ZM55.28a.sosip_ ZM55.28aSOSIP, R6, 664 I201C/A433C d7324 1580 A683 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, StabilityE168K, BG505 gp41 chim, chimera Y191W I201C-A433C, Y191W 1581 A684JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664 E168K, Y191WStability E168K, Y191W chimera 1582 A685 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, VRC26 binding chimeraR315Q E168K, BG505 gp41 chim, I201C-A433C, R315Q 1583 A686JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, VRC26 binding E168K, BG505 chimera residues 161-170gp41 chim, From CAP256 SU strain I201C-A433C, 161-170SU 1584 A687JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, VRC26 binding E168K, BG505 chimera residues 161-170gp41 chim, I201C-A433C, From CAP256 SU strain, 161-170SU, R313Q R313Q1585 A688 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, R315Q VRC26 binding E168K, R315Q chimera 1586 A689JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, residues 161-170 VRC26 binding E168K, 161-170, SUstrand C chimerafrom CAP256 SU strain 1587 A690 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505SOSIP, R6, 664 E168K, residues 161-170 VRC26 bindingE168K, 161-170SU, R313Q chimera from CAP256 SU strain, R313Q 1588 A691JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, Interprotomer DS E168K, BG505 gp41 chim, chimeraT128C, D167C I201C-A433C, T128C, D167C 1589 A692 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, Interprotomer DSE168K, BG505 gp41 chim, chimera V127C, D167C I201C-A433C, V127C, D167C1590 A693 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, T128C, D167C Interprotomer DS E168K, T128C, D167C chimera 1591A694 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, V127C, D167C Interprotomer DS E168K, V127C, D167C chimera 1592A695 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, Interprotomer DS E168K, BG505 gp41 chim, chimeraI165C, C196S I201C-A433C, I165C, C196S 1593 A696 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, Interprotomer DSE168K, BG505 gp41 chim, chimera I165C, C196F I201C-A433C, I165C, C196F1594 A697 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, Interprotomer DS E168K, BG505 gp41 chim, chimeraI165C, C196L I201C-A433C, I165C, C196L 1595 A698 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I165C, C196S Interprotomer DSE168K, ll65C, C196S chimera 1596 A699 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I165C, C196F Interprotomer DSE168K, ll65C, C196F chimera 1597 A700 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I165C, C196L Interprotomer DSE168K, ll65C, C196L chimera 1598 A701 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, DDSE168K, BG505 gp41 chim, chimera R304C/R440C I201C-A433C, R304C/R440C1599 A702 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, DDS E168K, BG505 gp41 chim, chimera A174C/T319CI201C-A433C, A174C/T319C 1600 A703 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, DDSE168K, BG505 gp41 chim, chimera S164C/H308C I201C-A433C, S164C/H308C1601 A704 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, I201C-A433C, Interprotomer DS E168K, BG505 gp41 chim, chimeraS110C/L556C I201C-A433C, S110C/L556C 1602 A705 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, S110C/L556C Interprotomer DSE168K, S110C/L556C chimera 1603 A706 JRFLgp140.6R.SOSIP.664.JRFLgp140/BG505 SOSIP, R6, 664 E168K, I201C-A433C, Interprotomer DSE168K, BG505 gp41 chim, chimera A558C/D113C I201C-A433C, A558C/D113C1604 A707 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, A558C/D113C Interprotomer DS E168K, A558C/D113C chimera 1605 A708JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664 E168K, E561W CavE168K, E561W chimera 1606 A709 JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505SOSIP, R6, 664 E168K, E561F Cav E168K, E561F chimera 1607 A710JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, E561W/K121F Cav E168K, E561W/K121F chimera 1608 A711JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, E561F/K121F Cav E168K, E561F/K121F chimera 1609 A712JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, E561W/K121W Cav E168K, E561W/K121W chimera 1610 A713JRFLgp140.6R.SOSIP.664. JRFLgp140/BG505 SOSIP, R6, 664E168K, E561F/K121W Cav E168K, E561F/K121W chimera 1611 A714BG505gp140.6R.SOSIP.664. BG505 SOSIP, R6, 664 T332N.D7325 Q315CT332N.D7325 Q315C 1612 A715 BG505gp140.6R.SOSIP.664. BG505SOSIP, R6, 664 F332N.D7324 V120C T332N.D7324_V120C 1613 A716JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K Q315C E168K_Q315C 1614A717 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168KV120C E168K_V120C1615 A718 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K E168K 1616A719 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K I201C/A433C DSE168K_I201C/A433C 1617 A720 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K A433P Stability E168K_A433P 1618 A721 JRFLgp140.6R.SOSIP.664. JRFLSOSIP, R6, 664 E168K_Q432P Stability E168K_Q432P 1619 A722JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K S174C/A319CE168K_S174C/A319C 1620 A723 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K N195C/A433C E168K_N195C/A433C 1621 A724 JRFLgp140.6R.SOSIP.664.JRFL SOSIP, R6, 664 E168K S199C/A433C E168K_S199C/A433C 1622 A725JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K R304C/Q440CE168K_R304C/Q440C 1623 A726 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K F223W Cav E168K_F223W 1624 A727 JRFLgp140.6R.SOSIP.664. JRFLSOSIP, R6, 664 E168K G473Y Cav E168K_G473Y 1625 A728JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K G431P StabilityE168K_G431P 1626 A729 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K N425C A433C E168K_N425C_A433C 1627 A730 JRFLgp140.6R.SOSIP.664.JRFL SOSIP, R6, 664 E168K V120C Q315C E168K_V120C_Q315C 1628 A731JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K Q203C L122CE168K_Q203C_L122C 1629 A732 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K_I201C/A433C/ E168K_I201C/A433C/R304 R304C/Q440C C/Q440C 1630 A733JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K R304C/R440CE168K_R304C/R440C 1631 A734 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K Q203C/F317C E168K_Q203C/F317C 1632 A735 JRFLgp140.6R.SOSIP.664.JRFL SOSIP, R6, 664 E168K L122C/F317C E168K_L122C/F317C 1633 A736JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K P437C/Y318CE168K_P437C/Y318C 1634 A737 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K E172C/I307C E168K_E172C/I307C 1635 A738 JRFLgp140.6R.SOSIP.664.JRFL SOSIP, R6, 664 E168K P206C/Y318C E168K_P206C/Y318C 1636 A739JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K A174C/T319CE168K_A174C/T319C 1637 A740 JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664E168K S164C/H308C E168K_S164C/H308C 1638 A741 JRFLgp140.6R.SOSIP.664.JRFL SOSIP, R6, 664 E168K T320C/L175C E168K_T320C/L175C 1639 A742JRFLgp140.6R.SOSIP.664. JRFL SOSIP, R6, 664 E168K T320C/P438CE168K_T320C/P438C 1640 A743 JRFLgp140.6R.SOS.664. JRFL 6R.SOS.664 E168Kstability E168K 1641 A744 JRFLgp140.6R.IP.664. JRFL 6R.IP.664 E168Kstability E168K 1642 A745 JRFLgp140.6R.664. JRFL 6R.664 E168K stabilityE168K 1643 B192 *703010505.TF-chim-sc_ 703010505.TF/BG505SOSIP, sc15ln, 664 I201C/A433C d7324, R6, 664, chimera I201C/A433CAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNATASNSSIIEGMKNCSFNITTELRDKREKKNALFYKLDIVQLDGNSSQYRLINCNTSVITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITNNVKTIIVHLNESVKIECTRPNNKTRTSIRIGPGQAFYATGQVIGDIREAYCNINESKWNETLQRVSKKLKEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTETNSTRTITIHCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDGGKNNTETFRPGGGNMKDNWRSELYKYKVVKEPLGVAPTRCKRRVVGggsggggsggggsggAVGIGAVFLGFLGAAGSTMGAASMTLTV0ARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1644 B193703010505.TF-chim- 703010505.TF/ SOSIP, I201C/A433C/ single chain 15sc_d7324_tad, BG505 sc15ln 664 E47D/K49E/ amino acid linker,sc15ln, 664, chimera V65K/E106T/E trimer association I201C/A433C/E47D/429R/R4320/ domain mutations K49E/V65K/E106T/ E500R E429R/R432 Q/E500R1645 B194 bg505.sosip_ BG505 SOSIP; 508(REKR)511 increase CD4-cl5ln.DS_gly3 664; replaced by binding site 201C, 433C linker ofaccessibility ength 15, 278A, oy removing 365A, 464A glycans around it1646 B195 bg505.sosip_ BG505 SOSIP; 508(REKR)511 Same as Seq_1645cl5ln.DS_gly4 664; replaced by 201C, 433C linker of ength 15, S199A,278A, 365A, 464A 1647 B196 bg505.sosip_ BG505 SOSIP; 508(REKR)511Same as Seq_1645 cl5ln.DS_gly5 664; replaced by 201C, 433C linker ofength 15, S199A, 278A, 365A, 388A, 464A 1648 B197 ZM55.28a-chim-ZM55/BG505 SOSIP; 5eq_0534 linker Same as Seq_1645 sc_DS_gly3 chimera664; between 508-511, 201C, 433C BG505 Platform, BG505 Res. Set B,remainder = ZM55.28a, 278A, 463A, 467A 1649 B198 ZM55.28a-chim-ZM55/BG505 SOSIP; Seq_0534 linker Same as Seq_1645 sc_DS_gly4 chimera664; between 508-511, 201C, 433C BG505 Platform, BG505 Res. Set B,remainder = ZM55.28a, S199A, 278A, 463A, 467A 1650 B199 ZM55.28a-chim-ZM55/BG505 SOSIP; Seq_0534 linker Same as Seq_1645 sc_DS_gly5 chimera664; between 508-511, 201C, 433C BG505 Platform, BG505 Res. Set B,remainder = ZM55.28a, S199A, 278A, 388A, 463A, 467A 1651 D011BG505sosip_ BG505 SOSIP, R6, 664, 278A, 365A, 464A Same as Seq_1645ig_I201C/ 201C/433C A433C.STOP-gly3 1652 D012 BG505sosip_ BG505SOSIP, R6, 664, S199A, 278A, Same as Seq_1645 ig_ 201C/433C 365A, 464AI201C/A433C.STOP-gly4 1653 D013 BG505sosip_ BG505 SOSIP, R6, 664,5199A, 278A, 365A, Same as Seq_1645 ig_ 201C/433C 388A, 464AI201C/A433C.STOP-gly5 1654 D014 CH505SOSIP_ CH505 SOSIP, R6, 664,278A, 463A Same as Seq_1645 DS_degly3 201C/433C 1655 D015 CH505SOSIP_CH505 SOSIP, R6, 664, 199A, 278A, 463A Same as Seq_1645 DS_degly4201C/433C 1656 D016 CH505SOSIP_ CH505 SOSIP, R6, 664,199A, 278A, 388A, 463A Same as Seq_1645 DS_degly5 201C/433C 1657 D017ZM55.28a-chim_ ZM55/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 DS_gly3 chimera 201C/433C BG505 Res. Set B,remainder = ZM55.28a, 278A, 463A, 467A 1658 D018 ZM55.28a-chim_ZM55/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 DS_gly4chimera 201C/433C BG505 Res. Set B, remainder = ZM55.28a, S199A, 278A,463A, 467A 1659 D019 ZM55.28a-chim_ ZM55/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1645 DS_gly5 chimera 201C/433CBG505 Res. Set B, remainder = ZM55.28a, S199A, 278A, 388A, 463A, 467A1660 D020 ZM106.9.sosip_ Zml096.9 SOSIP, R6, 664, 278A, 466ASame as Seq_1645 DS_gly3 201C/433C 1661 D021 ZM106.9.sosip_ Zml096.9SOSIP, R6, 664, S199A, 278A, 466A Same as Seq_1645 DS_gly4 201C/433C1662 D022 ZM106.9.sosip_ Zml096.9 SOSIP, R6, 664,S199A, 278A, 388A, 466A Same as Seq_1645 DS_gly5 201C/433C 1663 D023CH038.12-chim_ CH038/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 DS_gly3 chimera 201C/433C BG505 Res. Set B,remainder = CH038, 278A, 464A, 467A 1664 D024 CH038.12-chim_ CH038/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 DS_gly4 chimera201C/433C BG505 Res. Set B, remainder = CH038, 199A, 278A, 464A, 467A1665 D025 CH038.12-chim_ CH038/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 DS_gly5 chimera 201C/433C BG505 Res. Set B,remainder = CH038, 199A, 278A, 388A, 464A, 467A 1666 D026 ZM106.9-chim_ZM106/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 DS_gly3chimera 201C/433C BG505 Res. Set B, remainder = ZM106.9, 278A, 466A 1667D027 ZM106.9-chim_ ZM106/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 DS_gly4 chimera 201C/433C BG505 Res. Set B,remainder = ZM106.9, S199A, 278A, 466A 1668 D028 ZM106.9-chim_ZM106/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 DS_gly5chimera 201C/433C BG505 Res. Set B, remainder = ZM106.9, S199A, 278A,388A, 466A 1669 D029 16055-2.3-chim_ 10655/BG505 SOSIP, R6, 664,BG505 Platform, Same as Seq_1645 DS_gly3 chimera 201C/433CBG505 Res. Set B, remainder = 10655, 278A, 465A 1670 D03016055-2.3-chim_ 10655/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 DS_gly4 chimera 201C/433C BG505 Res. Set B,remainder = 10655, S199A, 278A, 465A 1671 D031 16055-2.3-chim_10655/BG505 SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 DS_gly5chimera 201C/433C BG505 Res. Set B, remainder = 10655,S199A, 278A, 388A, 465A 1672 D032 45_01dG5_bg505- 45-01dG5/BG505SOSIP, R6, 664, BG505 Platform, Same as Seq_1645 NCgp120 + gp41. chimera201C/433C BG505 Res. Set B, SOSIP_ remainder = 45 01dG5, DS_gly3 364A1673 D033 45_01dG5_bg505- 45-01dG5/BG505 SOSIP, R6, 664, BG505 Platform,Same as Seq_1645 NCgp120 + gp41. chimera 201C/433C BG505 SOSIP_Res. Set B, DS_gly4 remainder = 45 01dG5, S199A, 364A 1674 D034426c_degly3DS 426c SOSIP, R6, 664, 278A, 462A Same as Seq_1645 201C/433C1675 D035 426c_degly4DS 426c SOSIP, R6, 664, S199A, 278A, 462ASame as Seq_1645 201C/433C 1676 H385 *426c-v1v2- 426c/WITO- SOSIP, BG505WITO-degly4- Vlv2/BG505 R6, 664, chimera, S199A/I201C/A433C/ chimera5199A/I201C/ V1V2 chimera, N276D/N460D/ A433C/N276 glycan N463D/R504N/D/N460D/N463D/ shielding at V506T/L661N/ R504N/ residues 504, 661 L663TV506T/L661N/ L663TAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICaNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1677 H386 *426c-v1v2- 426c/WITO- SOSIP, BG505 WITO-degly3-Vlv2/BG505 R6, 664, chimera, I201C/A433C/ chimera I201C/A433C/V1V2 chimera, N276D/N460D/ N276D/N46 glycan N463D/R504N/ DD/N463D/shielding at V506T/ R504N/V506T/ residues L661N/L663T L661N/L663T504, 662AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTsTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1678 H387 *426c-v1v2- 426c/ZM233- SOSIP, BG505ZM233-degly4- Vlv2/BG505 R6, 664, chimera, S199A/I201C/ chimeraS199A/I201C/ V1V2 chimera, A433C/N276D/ A433C/N276D/ glycan N460D/N463DN460D/N463D/ shielding at /R504N/ R504N/ residues V506T/L661N/ V506T/504, 663 L663T L661N/L663TAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1679 H388 *426c-v1v2- 426c/ZM233- SOSIP,BG505 ZM233-degly3- Vlv2/BG505 R6, chimera, I201C/A433C/ chimera 664,V1V2 chimera, N276D/N460D/ I201C/ glycan N463D/R504N/ A433C/shielding at V506T/ N276D/ residues L661N/L663T N46DD/ 504, 664 N463D/R504N/ V506T/ L661N/ L663TAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICaNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1680 H389 *d45-01dG5chim- donor45- SOSIP,BG505 v1v2-WITO- 01dG5/ R6, 664, chimera, I201C/A433C/ WITO- I201C/V1V2 chimera, R504N/V506T/ V1V2/BG505 A433C/ glycan L661N/L663T chimeraR504N/ shielding at V506 residues 504, T/L661N/ 665 L663TAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTQQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1681 H390 *d45-01dG5chim- donor45- SOSIP,BG505 v1v2-WITO- 01dG5/WITO- R6, 664, chimera, 01dG5chim-degly3-V1V2/BG505 I201C/ V1V2 chimera, I201C/S364A/ chimera S364A/A433C/ glycanA433C/R504N/ R504 shielding at V506T/L661N/L663T N/V506T/ residuesL661N/L663T 504, 666AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFVKLDIVPIEGKNTNTGYRLINCNT5VCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPP1KGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQHYGLLEESQNQQEKNEQDLnAtD 1682 H391*d45-01dG5chim- donor45- SOSIP, BG505 v1v2-WITO- 01dG5/WITO- R6,chimera, 01dG5chim-degly4- V1V2/BG505 664, V1V2 chimera, S199A/I201C/chimera 5199A/ glycan S364A/A433C/ I201C/ shielding at R504N/V506T/S364A/ residues L661N/L A433C/ 504, 667 663T R504N/ V506T/ L661N/L6 S3TAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVaQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1683 H392 *d45-01dG5chim- donor45- SOSIP,BG505 v1v2-ZM233- 01dG5/ZM233- R6, 664, chimera, I201C/A433C/R504N/V1v2/BG505 I201C/A433C/ V1V2 chimera, V506T/L661N/L663T chimeraR504N/V506T glycan /L661N/L663T shielding at residues 504, 668AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1684 H393 *d45-01dG5chim- donor45-01dG5/ZM233- SOSIP, BG505v1v2-ZM233-degly3- V1v2/BG505 R6, chimera, I201C/S364A/A433C chimera664, V1V2 chimera, /R504N/V506T/ I201C/ glycan L661N/L663T S364A/shielding at A433C/ residues R504N/ 504, 669 V506T/ L661N/L663TAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1685 H394 *d45-01dG5chim-donor45- SOSIP, R6, BG505 v1v2-ZM233-degly4- 01dG5/ZM233- 664, chimera,S199A/201C/S364A/ V1V2/BG505 S199A/201C/ V1V2 chimera,A433C/R504N/V506T/ chimera S364A/A433 glycan L661N/L663T C/R504N/shielding at V506T/L661N/ residues L6S3T 504, 670AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTaVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQJRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 1686 H395 *426c-v1v2-426c/WITO- SOSIP, R6, BG505 WITO-degly4-  V1V2/BG505 664, chimera,S199A/I201C/A433C/ chimera S199A/I201C/ V1V2 chimera, N276D/N460D/N463DA433C/N276D/ glycan N460D/N463D shielding at residues 504, 661AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISVYTQIIYGLLEESQNQQEKNEQDLLALD 1687 H396 *426c-v1v2- 426c/WITO- SOSIP,BG505 WITO-degly3-  V1V2/BG505 R6, chimera, I201C/A433C/ chimera 664,V1V2 chimera, N276D/N460D/N463D I201C/ glycan A433C/ shielding at N276D/residues 504, N460D/ 662 N463DAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTsTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1688 H397 *426c-v1v2- 426c/ZM233-SOSIP=, BG505 ZM233-degly4- Vlv2/BG505 R6, chimera, S199A/I201C/A433C/chimera 664, V1V2 chimera, N276D/N460D/N463D 5199A/I201C/ glycanA433C/N276 shielding at D/N460D/ residues 504, 663 N463DAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1689 H398 *426c-v1v2- 426c/ZM233- SOSIP, BG505ZM233-degly3- Vlv2/BG505 R6, chimera, I201C/A433C/ chimera 664,V1V2 chimera, N276D/N460D/N463D I201C/A433C/ glycan N276D/N460D/shielding at N463D residues 504, 664AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTsTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1690 H399 *d45-01dG5chim- donor45- SOSIP,BG505 v1v2-WITO-I201C/ 01dG5/ R6, chimera, A433C WITO- 664,V1V2 chimera, V1V2/BG505 I201C/A433C glycan chimera shielding atresidues 504, 665AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIR0AHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1691 H400*d45-01dG5chim- donor45- SOSIP, R6, BG505 v1v2-WITO-degly3- 01dG5/WITO-664, chimera, I201C/S364A/A433C V1V2/BG505 I201C/S364A/ V1V2 chimera,chimera A433C glycan shielding at residues 504, 666AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIR0AHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1692 H401*d45-01dG5chim- donor45- SOSIP, R6, BG505 v1v2-WITO-degly4- 01dG5/WITO-664, chimera, S199A/I201C/S364A V1v2/BG505 S199A/I201C/V1V2 chimera, glycan chimera S364A/A433C shielding at residues 504, 667AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTaVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1693 H402*d45-01dG5chim-v1v2- donor45-01dG5/ SOSIP, R6, 664, BG505ZM233-l201C/A433C ZM233- I201C/A433C chimera, V1V2/BG505 V1V2 chimera,chimera glycan shielding at residues 504, 668AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1694 H403 *d45-01dGSchim-donor45- SOSIP, R6, BG505 v1v2-ZM233-degly3- 01dG5/ZM233- 664, chimera,I201C/S364A/A433C V1V2/BG505 I201C/ V1V2 chimera, chimera S364A/A433Cglycan shielding at residues 504, 669AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1695 H404 *d45-01dG5chim-donor45- SOSIP, R6, BG505 v1v2-ZM233-degly4- 01dG5/ZM233- 664, chimera,S199A/201C/ V1v2/BG505 S199A/201C/ V1V2 chimera, S364A/A433C chimeraS364A/A4330 glycan shielding at residues 504, 670AENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTaVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1696 H405 *ZM106.9-chim_ZM106.9/BG505 V1V2SOSIP; V1V2 Swap KER2018.11, DS_THS_ chimera 664;gly504-661 v1v2-KER2018.11- R6; 201C/D368R/433C/R504T/ 201C,V506T/L661N/L663T D368R, 433C, glycan504, glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCINANVTNSSMTNSSMMEGEIKNCSYNMTTELRDKKRKVFSLFYKLDVVPMNENNSEYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1697 H406 *ZM106.9-chim_ ZM106.9/BG505V1V2 V1V2 Swap CH070.1, DS_ chimera SOSIP; gly504-661 THS_v1v2-CH070.1-664; 201C/D368R/433C/ R6; R504T/V506T/L661N/ 201C, L663T D368R,433C, glycan504, glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLKCKDVSINNGNVSSSNGSTSHNNSSIDNETLNEGMKEMKNCSFVATTVLRDKKQKVHALFYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1698 H407 *ZM106.9-chim_ZM106.9/BG505 V1V2SOSIP; V1V2 Swap ZM233.6, DS_THS_ chimera 664;gly504-661 v1v2-ZM233.6- R6; 201C/D368R/433C/R504T/ 201C, D368R,V506T/L661N/L663T 433C, glycan504, glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1699 H408 *ZM106.9-chim_ ZM106.9/BG505V1V2SOSIP; V1V2 Swap Q23, DS_THS_ chimera 664; gly504-661 v1v2-Q23- R6;201C/D368R/433C/V506T/ 201C, R504T/L661N/L663T D368R, 433C, glycan504,glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHG'NVTSVNTTGDREGLKNCSFNMTTELRDKRQKVYSLFYRLDIYPINENQGSEYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNrTGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQaHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICaNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESaNQQEKNEQDLNATD 1700 H409 *ZM106.9-chim_ ZM106.9/BG505V1V2SOSIP; V1V2 Swap A244, DS_ chimera 664; gly504-661 THS_ v1v2-A244-R6; 201C/D368R/433C/ 201C, R504T/V506T/L661N/ D368R, 433C, L663Tglycan504, glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNANLTKANLTNVNNRTNVSNIIGNITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVPIEDNNDNSKYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1701 H410 *ZM106.9-chim_ZM106.9/BG505 V1V2SOSIP; V1V2 Swap WHO, gly504-661 DS_ chimera 664;THS_ v1v2-WITO- R6; 201C/D368R/433C/ 201C, R504T/V506T/L661N/ 368R,L663T 433C, glycan504, glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1702 H411 *ZM106.9-chim_ ZM106.9/BG505V1V2SOSIP; V1V2 Swap DS_THS_ chimera 664; CAP256-SU, v1v2-Cap256- R6;gly504-661 SU-201C/ 201C, D368R, 433C, D368R/433C/R504T/ glycan504,V506T/L661N/L663T glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCSDAKVNINATYNGTREEIKNCSFNATTELRDKKKKEYALFYRLDIVPLNKEGNNNSEYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDOQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1703 H412 *ZM106.9-chim_ ZM106.9/BG505 V1V2SOSIP;V1V2 Swap T250.4, DS_THS_ chimera 664; gly504-661 v1v2-T250.4- R6;201C/D368R/433C/R504T/ 201C,D368R, V506T/L661N/L663T 433C, glycan504,glycan661AENLWVTVYYGVPVWKEAKTTLFCASDAKSYEREVHNVWATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLDCQAFNSSSHTNSSIAMQEMKNCSFNVTTELRDKKKKEYSFFYKTDIEQINKNGRQYRLISCNTSTCTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLNATD 1704 H413 *ZM106.9-chim_ ZM106.9/BG505V1V2SOSIP; V1V2 Swap BB201.B42, DS_THS_ chimera 664; gly504-661v1v2-BB201.B42- R6; 201C, 201C/D368R/433C/R504T/ D368R, 433C,V506T/L661N/L663T glycan504, glycan661KTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYATGDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGRLEVTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRCMYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYT0IIYGLLEESQNQQEKNEQDLNATD 1705 H414 *cap256-su-chim_ cap256-suSOSIP, R6, 664, 201C/433C ds_THS-AviAENLWVTVYYGVPVWREAKTTLFCASDAKSYEKEVHNVWATHACVPTDPNPQELVLKNVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCSDAKVNINATYNGTREEIKNCSFNATTELRDKKKKEYALFYRLDVPLNKEGNNNSEYRLINCNTSVCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNVKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIR0AHCNSEIKWEKTLQRVSEKLREHFNKTIIFNQsSGGDLEITTHSFNCGGEFFYCNTSDLFFNKTFDETYSTGSNSTNSTITLPCRIKQIINMWQEVGRCMYASPIAGEITCKSNITGLLLTRDGGGNNSTEETFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1706 H415 *cap256-su-chim +cap256-su SOSIP, R6, 664, Interacting residues int_ 201C/433Cbetween the gp41 ds_THS-Avi and gp120AENLWVTVYYGVPVWKDAETTLFCASDAKSYEKEVHNVWATHACVPTDPNPQEIHLENVTENFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLNCSDAKVNINATYNGTREEIKNCSFNATTELRDKKKKEYALFYRLDIVPLNKEGNNNSEYRLINCNTSVCTQACPKVTFDPIPIHYCAPAGFAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNVKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEIKWEKTLQRVSEKLREHFNKTIIFNQsSGGDLEITTHSFNCGGEFFYCNTSDLFFNKTFDETYSTGSNSTNSTITLPCRIKQIINMWQEVGRCMYASPIAGEITCKSNITGLLLTRDGGGNNSTEETFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1707 H416*426c-native_bg505- 426c/BG505 SOSIP; NCgp120 + gp41. chimera 664; SOSIPR6 AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLSDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIW 1708H417 *426c-del276-460_bg505- 426c/BG505 SOSIP; removal of glycan 276NCgp120 + gp41. chimera 664; SOSIP R6AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGNTaNNTEIFRPGGGDMRDNWRSELYKYKVVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1709 H418 *426c-del276 bg505-426c/BG505 SOSIP; removal of glycan 276 NCgp120 + gp41. chimera 664SOSIP AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1710 H419 *426c-del276-460 DS426c/BG505 SOSIP, R6, 664, removal of glycan 276 bg505- chimera201C/433C and glycan 460 NCgp120 + gp41. SOSIPAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTaNNTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1711 H420 *426c-de1276 DS bg505-426c/BG505 SOSIP, R6, removal of glycan 276 NCgp120 + gp41. chimera 664,SOSIP 201C/433CAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEmHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1712 H421 *426c-native DS bg505-426c/BG505 SOSIP, R6, 664, NCgp120 + gp41. chimera 201C/433C SOSIPAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLSDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEmHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD H422 *426c-d3GLY-SUstrC bg505-426c/BG505 SOSIP; Introduction of cap256 NCgp120 + gp41. chimera 664;su strand c SOSIP R6 1713AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFnatte1rdkkkKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPP1KGNITCKSDITGLLLLRDGGNTaNNaEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD H423 *426c-d3GLY- 426c/BG505SOSIP, R6, 664, Introduction of cap256 SUstrC_DS bg505- chimera201C/433C su strand c NCgp120 + gp41. SOSIP 1714AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFnattelrdkkkKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSWQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLaDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTaNNaEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD H424 *703010505.TF-chim_703010505.TF/BG505 SOSIP, R6, 664 I201C/A433C d7324, R6, 664, chimera1201C/A433C 1715AENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMHEDVISLWDQSLKPCVKLTPLCmNCTNATASNSSIIEGMKNCSFNITTELRDKREKKNALFYKLDIVQLDGNSSQYRLINCNTSVITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITNNVKTIIVHLNESVKIECTRPNNKTRTSIRIGPGQAFYATGQVIGDIREAYCNINESKWNETLQRVSKKLKEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTETNSTRTITIHCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDGGKNNTETFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1716 H425 703010505.TF-chim + int_703010505.TF/BG505 SOSIP, R6, 664 I201C/A433C interface mutationsd7324, R6, 664, chimera I201C/A433C 1717 H426 703010505.TF_cl-703010505.TF/BG505 SOSIP, R6, 664 I201C/A433C small-sel_d7324, chimeraR6, 664, I201C/A433C 1718 H427 703010505.TF_cl1- 703010505.TF/BG505SOSIP, R6, 664 I201C/A433C 2_d7324, R6, 664, chimera I201C/A433C 1719H428 703010505.TF_cll_ 703010505.TF/BG505 SOSIP, R6, 664 I201C/A433Cd7324, R6, 664, chimera I201C/A433C 1720 H429 703010505.TF-chim_703010505.TF/BG505 SOSIP, R6, 664 I201C/A433C trimer associationd7324_tad, R6, 664, chimera domain mutations I201C/A433C/E47D/K49E/V65K/E106T/ E429R/R432Q/ E500R 1721 H430 703010505.TF_cl-703010505.TF/BG505 SOSIP, R6, 664 I201C/A433C trimer associationsmall-sel_d7324_ chimera domain mutations tad, R6, 664,I201C/A433C/E47D/ K49E/V65K/E106T/ E429R/R432 Q/E500R 1722 H431703010505.TF_cll-2_ 703010505.TF/BG505 SOSIP, R6, 664 I201C/A433Ctrimer association d7324_tad, R6, 664, chimera domain mutationsI201C/A433C/E47D/K49E/ V65K/E106T/E429R/ R432Q/E500R 1723 H432703010505.TF_cll_ 703010505.TF/ SOSIP, R6, 664 I201C/A433Ctrimer association d7324_tad, R6, 664, BG505 domain mutations chimeraI201C/A433C/E47D/K49E/ V65K/E106T/E429R/R432 Q/E500R 1724 H433703010505.TF-chim + int_ 703010505.TF/BG505 SOSIP, R6, 664 I201C/A433Cinterface mutations, d7324_tad, R6, 664, chimera trimerI201C/A433C/E47D/K49E/ association domain V65K/E106T/E429R/R432Q/mutations E500R 1725 H434 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168K, DS E168K, BG505 gp41 chim, I201C, I201C, A433C,A433C, 1726 H435 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, E168K, BG505 gp41 chim, + interface +int I201C, A433C, residues,I201C, A433C, 1727 H436 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168K, , V2 from BG505 E168K, BG505 gp41 chim, I201C,I201C, A433C, v2 A433C, _BG505 V2 (residues 154-205), 1728 H437JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664 E168K, ,V1, 191-205 from BG505 E168K, BG505 gp41 chim, I201C,I201C, A433C, vl 191-205 A433C, _BG505 VI (residues 119-153), 191-2051729 H438 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, , V1 from BG505 E168K, BG505 gp41 chim, + interface+int I201C, A433C, v1 residues, I201C, A433C,_BG505 V1 (residues119-153), 1730 H439 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168K, , 166-173 from BG505 E168K, BG505 gp41 chim,I201C, I201C, A433C, strC A433C, _strC 1731 H440 JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, , 166-173 from BG505E168K, BG505 gp41 chim, + interface +int I201C, A433C, strC residues,I201C, A433C, _strC 1732 H441 *JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168K, V2, V3 from BG505 E168K, BG505 gp41 chim, I201C,I201C, A433C,_v2_v3 A433C, _BG505 V2 (residues 154-205), _BG505 V3(residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1733 H442JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, 191-205, 166-173 E168K, BG505 gp41 chim,_BG505 V1 from BG505 I201C, A433C, _v1_strC_191-205 (residues 119-153),_strC_191-205 1734 H443 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168K, , + interface V1, 166-173 from BG505E168K, BG505 gp41 chim, residues, I201C, +int I201C, A433C, _v1_strCA433C, _BG505 V1 (residues 119-153), strC 1735 H444*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V3 from BG505 E168K, BG505 gp41 chim, _BG505 V3I201C, A433C, v3 (residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1736 H445 *JRFLgp140.6R.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface 166-173, SOSIP.664.residue set A, I201C, V3 from BG505 E168K, BG505 A433C, _strC_gp41 chim, BG505 V3 (residues 296- +int I201C, 331), A433C, _strC_v3AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKKQKVYALFYKLDVVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1737 H446 JRFLgp140.6R.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface V2 from BG505SOSIP.664. residues, I201C, E168K, BG505 A433C, BG505 gp41 chim,V2 (residues 154-205), +int I201C, A433C, v2 1738 H447 *JRFLgp140.6R.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface 191-205, SOSIP.664.residue set A, I201C, V3 from BG505 E168K, BG505 gp41 chim,A433C, _191-205_BG505 V3 +int I201C, (residues 296-331),A433C, _191-205_v3AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1739 H448 *JRFLgp140.6R.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface V1, V3 from BG505SOSIP.664. residue set A, I201C, E168K, BG505 A433C, _BG505 V1gp41 chim, (residues 119-153), +int BG505 V3 I201C, A433C, _v1_v3(residues 296-331),AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1740 H449 *JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, I201C, A433C, 166-173, V3E168K, BG505 gp41 chim, _strC_BG505 V3 from BG505 I201C, A433C, _strC_v3(residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKKQKVYALFYKLDVVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1741 H450*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface V3 from BG505 E168K, BG505 gp41 chim, residueHnt I201C, A433C, _v3 set A, I201C, A433C, _BG505 V3 (residues 296-331),AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1742 H451*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface V2, V3 from BG505 E168K, BG505 gp41 chim, residue+int I201C, set A, I201C, A433C, _v2_v3 A433C, _BG505 V2(residues 154-205), BG505 V3 (residues 296-331)AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1743 H452JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, v1, 166-173, 191-205, E168K, BG505 gp41 chim,_BG505 V1 V3 from I201C, A433C, _v1_ (residues 119-153), BG505strC_191-205_v3 _strC_191- 205 BG505 V3 (residues 296-331), 1744 H453*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface v1, 166-173, V3 E168K, BG505 gp41 chim,residue set A, I201C, from BG505 +int I201C, A433C, _v1_strC_v3A433C, _BG505 V1 (residues 119-153), strC BG505V3 (residues 296-331)AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGEIKNCSFNITTSIRDKKQKVYALFYKLDWPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1745 H454 JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, I201C, A433C, 191-205 from BG505E168K, BG505 gp41 chim, _191-205 I201C, A433C, 191-205 1746 H455JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface 191-205 from BG505 E168K, BG505 gp41 chim,residues, I201C, +int I201C, A433C, 191-205 A433C, 191-205 1747 H456JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, V2 from BG505 E168K, BG505 gp41 chim, _BG505 V1I201C, A433C, _v1_v2 (residues 119-153), _BG505 V2 (residues 154-205),1748 H457 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1 from BG505 E168K, BG505 gp41 chim, _BG505 V1I201C, A433C, vl (residues 119-153), 1749 H458 JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface V1, 191-205 from BG505E168K, BG505 gp41 chim, residue set A, I201C, +int I201C, A433C, _A433C, _BG505 V1 v1_191-205 (residues 119-153), 191-205 1750 H459JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, 166-173, 191-205 from BG505 E168K, BG505 gp41 chim,_strC_191-205 I201C, A433C, _strC_191-205 1751 H460JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface 166-173, 191-205 from BG505 E168K, BG505 gp41 chim.residue set A, I201C, +int I201C, A433C, A433C, strC 191-205StrC 191-205 1752 H461 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168KJ201C, A433C, V1, V2, V3 from BG505E168K, BG505 gp41 chim, _BG505 V1 (residues I201C, A433C, _v1_v2_v3119-153), _BG505 V2 (residues 154-205), BG505 V3 (residues 296-331)AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1753 H462JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, 166-173 from BG505 E168K, BG505 gp41 chim,_BG505 V1 (residues I201C, A433C, vl strC 119-153), strC 1754 H463JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface V1, 166-173, 191-205 from BG505E168K, BG505 gp41 chim, residue set A, I201C, +int I201C,A433C, _BG505 V1 A433C, _v1_strC_191-205 (residues 119-153),strC 191-205 1755 H464 JRFLgp140.6R.SOSIP.664. JRFL/BG505 chimSOSIP, R6, 664 E168KJ201C, A433C, 166-173, 191-205,E168K, BG505 gp41 chim, _strC_191-205 BG505 V3 V3 from BG505I201C, A433C, (residues 296-331), StrC 191-205 v3 1756 H465JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, 191-205, V3 from BG505 E168K, BG505 gp41 chim,_BG505 V1 (residues I201C, A433C, _v1_191-205_v3 119-153),_191-205_BG505 V3 (residues 296-331), 1757 H466 JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interface V1, V2 from BG505E168K, BG505 gp41 chim, residues, I201C, +int I201C, A433C, _v1_v2A433C, _BG505 V1 (residues 119-153), BG505 V2 (residues 154-205), 1758H467 *JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, V3 from BG505 E168K, BG505 gp41 chim,_BG505 V1 (residues I201C, A433C, _v1_v3 119-153), _BG505 V3(residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGEIKNCSFNITTSIRDKVQKEYALFYKLDWVPIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1759 H468 *JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, + interfaceV1, 191-205, V3 from BG505 E168K, BG505 gp41 chim, residues, I201C,+int I201C, A433C, A433C, _BG505 V1 _v1_191-205_v3 (residues 119-153),191-205 BG505 V3 (residues 296-331),AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1760 H469 *JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168KJ201C, A433C, 191-205, V3 from BG505E168K, BG505 gp41 chim, _191-205_BG505 V3 I201C, A433C, _191-205_v3(residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNNTSYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCVISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1761 H470*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface 166-173, 191-205, V3 from BG505E168K, BG505 gp41 chim, residues, I201C, +int I201C, A433C, _strC_191-A433C, _strC_191-205_v3 205_BG505 V3 +residues 296-331),AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDKKQKVYALFYKLDVVPIDNNNTSYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCVISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1762 4471*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, + interface V1, V2, V3 E168K, BG505 gp41 chim, residues, I201C,from BG505 +int I201C, A433C, _BG505 V1 A433C, _v1_v2_v3(residues 119-153), _BG505 V2 (residues 154-205), _BG505V3 (residues 296-331),AENLWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENVTEHFNMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQL0ARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1763 H472*JRFLgp140.6R.SOSIP.664. JRFL/BG505 chim SOSIP, R6, 664E168K, I201C, A433C, V1, 166-173, V3 from BG505 E168K, BG505 gp41 chim,_BG505 V1 I201C, A433C, _v1_strC_v3 (residues 119-153), _strC_BG505 V3(residues 296-331),AENLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGEIKNCSFNITTSIRDKKQKVYALFYKLDWVIDNNNTSYRLISCDTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKCMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD 1764 H473 JRFLgp140.6R.SOSIP.664.JRFL/BG505 chim SOSIP, R6, 664 E168K, + v1, 166-173,E168K, BG505 gp41 chim, interface residues, 191-205, V3 from+int I201C, A433C, I201C, BG505 _v1_strC_191-205_v3 A433C, _BG505V1 (residues 119-153), _strC_191-205_ BG505 V3 (residues 296-331), 1765T030 TH966.8-chim_ TH966.8/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera-10ln-HATM 1766 T031 6545.V4.C1-chim_ 5545.V4.C1/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1767 T032R2184.c4-chim_ R2184.c4/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera-10ln-HATM 1768 T033 ZM197.7-chim_ ZM197.7/BG505 sc10ln-IP-10ln-HATMsc10ln-IP chimera -10ln-HATM 1769 T034 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1770 T035 ZM53.12-chim_ZM53.12/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1771 T036R2184.c4-chim_ R2184.c4/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1772 T037 CNE55-chim_ CNE55/BG505 sc10ln-IP-10ln-HATM sc10ln-IPchimera -10ln-HATM 1773 T038 6545.V4.C1-chim_ 5545.V4.C1/BG505sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1774 T039 DU422.01-chim_U422.01/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1775 T04025925-2.22-chim_ 25925-2.22/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera-10ln-HATM 1776 T041 CNE58-chim_ CNE58/BG505 sc10ln-IP-10ln-HATMsc10ln-IP chimera -10ln-HATM 1777 T042 16055-2.3-chim_ 16055-2.3/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1778 T043 rH966.8-chim_rH966.8/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1779 T044ZM55.28a-chim_ ZM55.28a/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1780 T045 ZM53.12-chim_ ZM53.12/BG505 sc10ln-IP-MPER-TMsc10ln-IP chimera -MPER-TM 1781 T046 BI369.9A-chim_ BI369.9A/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1782 T047 ZM197.7-chim_ZM197.7/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1783 T04816055-2.3-chim_ 16055-2.3/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1784 T049 ZM55.28a-chim_ ZM55.28a/BG505 sc15ln-SOS-DS-10ln-HATMsc15ln-SOS chimera -DS-10ln-HATM 1785 T050 ZM106.9-chim_ ZM106.9/BG505SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1786 T051 AC10.29-chim_AC10.29/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1787 T052CH038.12-chim_ CH038.12/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM1788 T053 FRO.11-chim_ FRO.11/BG505 SOS-DS-10ln-HATM SOS chimera-DS-10ln-HATM 1789 T054 QH209.14M.A2-chim_ QH209.14M.A2/BG505SOS-DS-10ln-HATM SOS -DS-10ln-HATM chimera 1790 T055 6545.V4.C1-chim_5545.V4.C1/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1791 T056KER2018.11-chim_ KER2018.11/BG505 SOS-DS-10ln-HATM SOS chimera-DS-10ln-HATM 1792 T057 MB201.A1-chim_ MB201.A1/BG505 SOS-DS-10ln-HATMSOS chimera -DS-10ln-HATM 1793 T058 BI369.9A-chim_ BI369.9A/BG505SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1794 T059 CNE55-chim_CNE55/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1795 T060FH966.8-chim_ TH966.8/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM1796 T061 R2184.c4-chim_ 32184.C4/BG505 SOS-DS-10ln-HATM SOS chimera-DS-10ln-HATM 1797 T062 DU422.01-chim_ U422.01/BG505 SOS-DS-10ln-HATMSOS chimera -DS-10ln-HATM 1798 T063 16055-2.3-chim_ 16055-2.3/BG505SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1799 T064 ZM55.28a-chim_ZM55.28a/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1800 T065CH117.4-chim_ CH117.4/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM1801 T066 CNE58-chim_ CNE58/BG505 SOS-DS-10ln-HATM SOS chimera-DS-10ln-HATM 1802 T067 ZM53.12-chim_ ZM53.12/BG505 SOS-DS-10ln-HATM SOSchimera -DS-10ln-HATM 1803 T068 ZM197.7-chim_ ZM197.7/BG505SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1804 T069 25925-2.22-chim_25925-2.22/BG505 SOS-DS-10ln-HATM SOS chimera -DS-10ln-HATM 1805 T070ZM106.9-chim_ ZM106.9/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1806T071 AC10.29-chim_ AC10.29/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM1807 T072 CH038.12-chim_ CH038.12/BG505 SOS-DS-MPER-TM SOS chimera-DS-MPER-TM 1808 T073 TRO.11-chim_ TRO.11/BG505 SOS-DS-MPER-TM SOSchimera -DS-MPER-TM 1809 T074 QH209.14M.A2-chim_ QH209.14M.A2/BG505SOS-DS-MPER-TM SOS -DS-MPER-TM chimera 1810 T075 6545.V4.C1-chim_5545.V4.C1/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1811 T076KER2018.11-chim_ KER2018.11/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM1812 T077 MB201.A1-chim_ MB201.A1/BG505 SOS-DS-MPER-TM SOS chimera-DS-MPER-TM 1813 T078 BI369.9A-chim_ BI369.9A/BG505 SOS-DS-MPER-TM SOSchimera -DS-MPER-TM 1814 T079 CNE55-chim_ CNE55/BG505 SOS-DS-MPER-TM SOSchimera -DS-MPER-TM 1815 T056 rH966.8-chim_ FH966.8/BG505 SOS-DS-MPER-TMSOS chimera -DS-MPER-TM 1816 T081 R2184.c4-chim_ 32184.C4/BG505SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1817 T082 DU422.01-chim_U422.01/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1818 T08316055-2.3-chim_ 16055-2.3/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM1819 T084 ZM55.28a-chim_ ZM55.28a/BG505 SOS-DS-MPER-TM SOS chimera-DS-MPER-TM 1820 T085 CH117.4-chim_ CH117.4/BG505 SOS-DS-MPER-TM SOSchimera -DS-MPER-TM 1821 T056 CNE58-chim_ CNE58/BG505 SOS-DS-MPER-TM SOSchimera -DS-MPER-TM 1822 T087 ZM53.12-chim_ ZM53.12/BG505 SOS-DS-MPER-TMSOS chimera -DS-MPER-TM 1823 T088 ZM197.7-chim_ ZM197.7/BG505SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1824 T089 25925-2.22-chim_25925-2.22/BG505 SOS-DS-MPER-TM SOS chimera -DS-MPER-TM 1825 T090ZM106.9-chim_ ZM106.9/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1826 T091 AC10.29-chim_ AC10.29/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1827 T092CH038.12-chim_ CH038.12/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1828 T093 FRO.11-chim_ FRO.11/BG505 SOS-DS-MPER-TM-cytoSOS chimera -DS-MPER-TM-cyto 1829 T094 QH209.14M.A2-chim_QH209.14M.A2/BG505 SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1830T095 6545.V4.C1-chim_ 5545.V4.C1/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1831 T096 KER2018.11-chim_ KER2018.11/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1832 T097MB201.A1-chim_ MB201.A1/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1833 T098 BI369.9A-chim_ BI369.9A/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1834 T099 CNE55-chim_CNE55/BG505 SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1835 T100FH966.8-chim_ FH966.8/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1836 T101 R2184.c4-chim_ 32184.C4/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1837 T102DU422.01-chim_ U422.01/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1838 T103 16055-2.3-chim_ 16055-2.3/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1839 T104ZM55.28a-chim_ ZM55.28a/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1840 T105 CH117.4-chim_ CH117.4/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1841 T106 CNE58-chim_CNE58/BG505 SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1842 T107ZM53.12-chim_ ZM53.12/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1843 T108 ZM197.7-chim_ ZM197.7/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 1844 T10925925-2.22-chim_ 25925-2.22/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 1845 T110 AC10.29-chim_ AC10.29/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1846 T111CH038.12-chim_ CH038.12/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera-10ln-HATM 1847 T112 FRO.11-chim_ FRO.11/BG505 sc10ln-IP-10ln-HATMsc10ln-IP chimera -10ln-HATM 1848 T113 QH209.14M.A2-chim_QH209.14M.A2/BG505 sc10ln-IP-10ln-HATM sc10ln-IP -10ln-HATM chimera 1849T114 KER2018.11-chim_ KER2018.11/BG505 sc10ln-IP-10ln-HATM sc10ln-IPchimera -10ln-HATM 1850 T115 MB201.A1-chim_ MB201.A1/BG505sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1851 Tu6 ZM55.28a-chim_ZM55.28a/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera -10ln-HATM 1852T117 CH117.4-chim_ CH117.4/BG505 sc10ln-IP-10ln-HATM sc10ln-IP chimera-10ln-HATM 1853 T115 CNE58-chim_ CNE58/BG505 sc10ln-IP-10ln-HATMsc10ln-IP chimera -10ln-HATM 1854 T119 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1855 T120 AC10.29-chim_AC10.29/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1856 T121CH038.12-chim_ CH038.12/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1857 T122 FRO.11-chim_ FRO.11/BG505 sc10ln-IP-MPER-TM sc10ln-IPchimera -MPER-TM 1858 T123 QH209.14M.A2-chim_ QH209.14M.A2/BG505sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1859 T124 KER2018.11-chim_KER2018.11/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1860 T125MB201.A1-chim_ MB201.A1/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1861 T126 BI369.9A-chim_ BI369.9A/BG505 sc10ln-IP-MPER-TMsc10ln-IP chimera -MPER-TM 1862 T127 CNE55-chim_ CNE55/BG505sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1863 T128 DU422.01-chim_U422.01/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM 1864 T129CH117.4-chim_ CH117.4/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera -MPER-TM1865 T130 CNE58-chim_ CNE58/BG505 sc10ln-IP-MPER-TM sc10ln-IP chimera-MPER-TM 1866 T131 25925-2.22-chim_ 25925-2.22/BG505 sc10ln-IP-MPER-TMsc10ln-IP chimera -MPER-TM 1867 T132 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1868 T133AC10.29-chim_ AC10.29/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1869 T134 CH038.12-chim_ CH038.12/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1870 T135FRO.11-chim_ FRO.11/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1871 T136 QH209.14M.A2-chim_ QH209.14M.A2/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP -MPER-TM-cyto chimera 1872 T1376545.V4.C1-chim_ 5545.V4.C1/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IPchimera -MPER-TM-cyto 1873 T138 KER2018.11-chim_ KER2018.11/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1874 T139MB201.A1-chim_ MB201.A1/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1875 T140 BI369.9A-chim_ BI369.9A/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1876 T141CNE55-chim_ CNE55/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1877 T142 FH966.8-chim_ FH966.8/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1878 T143R2184.c4-chim_ 32184.C4/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1879 T144 DU422.01-chim_ U422.01/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1880 T14516055-2.3-chim_ 16055-2.3/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1881 T146 ZM55.28a-chim_ ZM55.28a/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1882 T147CH117.4-chim_ CH117.4/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1883 T148 CNE58-chim_ CNE58/BG505 sc10ln-IP-MPER-TM-cytosc10ln-IP chimera -MPER-TM-cyto 1884 T149 ZM53.12-chim_ ZM53.12/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1885 T150ZM197.7-chim_ ZM197.7/BG505 sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera-MPER-TM-cyto 1886 T151 25925-2.22-chim_ 25925-2.22/BG505sc10ln-IP-MPER-TM-cyto sc10ln-IP chimera -MPER-TM-cyto 1887 T152ZM106.9-chim_ ZM106.9/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1888 T153 AC10.29-chim_ AC10.29/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1889 T154CH038.12-chim_ CH038.12/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1890 T155 FRO.11-chim_ FRO.11/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1891 T156QH209.14M.A2-chim_ QH209.14M.A2/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOSchimera -DS-10ln-HATM 1892 T157 6545.V4.C1-chim_ 5545.V4.C1/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1893 T158KER2018.11-chim_ KER2018.11/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOSchimera -DS-10ln-HATM 1894 T159 MB201.A1-chim_ MB201.A1/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1895 T160BI369.9A-chim_ BI369.9A/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1896 T161 CNE55-chim_ CNE55/BG505 sc15ln-SOS-DS-10ln-HATMsc15ln-SOS chimera -DS-10ln-HATM 1897 T162 TH966.8-chim_ FH966.8/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1898 T163R2184.c4-chim_ 32184.C4/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1899 T164 DU422.01-chim_ U422.01/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1900 T16516055-2.3-chim_ 16055-2.3/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOSchimera -DS-10ln-HATM 1901 T166 CH117.4-chim_ CH117.4/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1902 T167CNE58-chim_ CNE58/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1903 T168 ZM53.12-chim_ ZM53.12/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1904 T169ZM197.7-chim_ ZM197.7/BG505 sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera-DS-10ln-HATM 1905 T170 25925-2.22-chim_ 25925-2.22/BG505sc15ln-SOS-DS-10ln-HATM sc15ln-SOS chimera -DS-10ln-HATM 1906 T171ZM106.9-chim_ ZM106.9/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1907 T172 AC10.29-chim_ AC10.29/BG505 sc15ln-SOS-DS-MPER-TMsc15ln-SOS chimera -DS-MPER-TM 1908 T173 CH038.12-chim_ CH038.12/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1909 T174TRO.11-chim_ FRO.11/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1910 T175 QH209.14M.A2-chim_ QH209.14M.A2/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1911 T1766545.V4.C1-chim_ 5545.V4.C1/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOSchimera -DS-MPER-TM 1912 T177 KER2018.11-chim_ KER2018.11/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1913 T178MB201.A1-chim_ MB201.A1/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1914 T179 BI369.9A-chim_ BI369.9A/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1915 T180CNE55-chim_ CNE55/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1916 T181 FH966.8-chim_ FH966.8/BG505 sc15ln-SOS-DS-MPER-TMsc15ln-SOS chimera -DS-MPER-TM 1917 T182 sd5ln-SOS 12184.C4/BG505sc15ln-SOS-DS-MPER-TM -DS-MPER-TM chimera 1918 T183 DU422.01-chim_U422.01/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1919T184 16055-2.3-chim_ 16055-2.3/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOSchimera -DS-MPER-TM 1920 T185 ZM55.28a-chim_ ZM55.28a/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1921 T186CH117.4-chim_ CH117.4/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1922 T187 CNE58-chim_ CNE58/BG505 sc15ln-SOS-DS-MPER-TMsc15ln-SOS chimera -DS-MPER-TM 1923 T188 ZM53.12-chim_ ZM53.12/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1924 T189ZM197.7-chim_ ZM197.7/BG505 sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera-DS-MPER-TM 1925 T190 25925-2.22-chim_ 25925-2.22/BG505sc15ln-SOS-DS-MPER-TM sc15ln-SOS chimera -DS-MPER-TM 1926 T191ZM106.9-chim_ ZM106.9/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimeracyto -DS-MPER-TM-cyto 1927 T192 AC10.29-chim_ AC10.29/8G505 chimerasc15ln-SOS-DS-MPER-TM- sc15ln-SOS cyto -DS-MPER-TM-cyto 1928 T193CH038.12-chim_ CH038.12/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimeracyto -DS-MPER-TM-cyto 1929 T194 TRO.11-chim_ TRO.11/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1930T195 QH209.14M.A2-chim_ QH209.14M.A2/BG505 sc15ln-SOS-DS-MPER-TM-sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1931 T196 6545.V4.C1-chim_6545.V4.C1/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto-DS-MPER-TM-cyto 1932 T197 KER2018.11-chim_ KER2018.11/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1933T198 MB201.A1-chim_ MB201.A1/8G505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1934 T199 BI369.9A-chim_ BI369.9A/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1935T200 CNE55-chim_ CNE55/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimeracyto -DS-MPER-TM-cyto 1936 T201 FH966.8-chim_ FH966.8/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1937T202 R2184.c4-chim_ 32184.C4/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1938 T203 DU422.01-chim_ U422.01/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1939T204 16055-2.3-chim_ 16055-2.3/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1940 T205 ZM55.28a-chim_ ZM55.28a/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1941T206 CH117.4-chim_ CH117.4/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1942 T207 CNE58-chim_ CNE58/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1943T208 ZM53.12-chim_ ZM53.12/BG505 sc15ln-SOS-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1944 T209 ZM197.7-chim_ ZM197.7/BG505sc15ln-SOS-DS-MPER-TM- sc15ln-SOS chimera cyto -DS-MPER-TM-cyto 1945T210 25925-2.22-chim_ 25925-2.22/BG505 sc15ln-S0S-DS-MPER-TM- sc15ln-SOSchimera cyto -DS-MPER-TM-cyto 1946 T211 ZM106.9-chim_ ZM106.9/BG505IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1947 T212 AC10.29-chim_AC10.29/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1948 T213CH038.12-chim_ CH038.12/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM1949 T214 FRO.11-chim_ FRO.11/BG505 IP-DS-10ln-HATM IP chimera-DS-10ln-HATM 1950 T215 QH209.14M.A2-chim_ QH209.14M.A2/BG505IP-DS-10ln-HATM IP -DS-10ln-HATM chimera 1951 T216 6545.V4.C1-chim_5545.V4.C1/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1952 T217KER2018.11-chim_ KER2018.11/BG505 IP-DS-10ln-HATM IP chimera-DS-10ln-HATM 1953 T218 MB201.A1-chim_ MB201.A1/BG505 IP-DS-10ln-HATM IPchimera -DS-10ln-HATM 1954 T219 BI369.9A-chim_ BI369.9A/BG505IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1955 T220 CNE55-chim_CNE55/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1956 T221FH966.8-chim_ TH966.8/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM1957 T222 R2184.c4-chim_ 32184.c4/BG505 IP-DS-10ln-HATM IP chimera-DS-10ln-HATM 1958 T223 DU422.01-chim_ U422.01/BG505 IP-DS-10ln-HATM IPchimera -DS-10ln-HATM 1959 T224 16055-2.3-chim_ 16055-2.3/BG505IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1960 T225 ZM55.28a-chim_ZM55.28a/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1961 T226CH117.4-chim_ CH117.4/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM1962 T227 CNE58-chim_ CNE58/BG505 IP-DS-10ln-HATM IP chimera-DS-10ln-HATM 1963 T228 ZM53.12-chim_ ZM53.12/BG505 IP-DS-10ln-HATM IPchimera -DS-10ln-HATM 1964 T229 ZM197.7-chim_ ZM197.7/BG505IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1965 T230 25925-2.22-chim_25925-2.22/BG505 IP-DS-10ln-HATM IP chimera -DS-10ln-HATM 1966 T231ZM106.9-chim_ ZM106.9/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM 1967T232 AC10.29-chim_ AC10.29/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM1968 T233 CH038.12-chim_ CH038.12/BG505 IP-DS-MPER-TM IP chimera-DS-MPER-TM 1969 T234 TRO.11-chim_ FRO.11/BG505 IP-DS-MPER-TM IP chimera-DS-MPER-TM 1970 T235 QH209.14M.A2-chim_ QH209.14M.A2/BG505IP-DS-MPER-TM IP -DS-MPER-TM chimera 1971 T236 6545.V4.C1-chim_5545.V4.C1/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM 1972 T237KER2018.11-chim_ KER2018.11/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM1973 T238 MB201.A1-chim_ MB201.A1/BG505 IP-DS-MPER-TM IP chimera-DS-MPER-TM 1974 T239 BI369.9A-chim_ BI369.9A/BG505 IP-DS-MPER-TM IPchimera -DS-MPER-TM 1975 T240 CNE55-chim_ CNE55/BG505 IP-DS-MPER-TM IPchimera -DS-MPER-TM 1976 T241 FH966.8-chim_ rH966.8/BG505 IP-DS-MPER-TMIP chimera -DS-MPER-TM 1977 T242 R2184.c4-chim_ 32184.c4/BG505IP-DS-MPER-TM IP chimera -DS-MPER-TM 1978 T243 DU422.01-chim_U422.01/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM 1979 T24416055-2.3-chim_ 16055-2.3/BG505 IP-DS-MPER-TM IP chimera -DS-MPER-TM1980 T245 ZM55.28a-chim_ ZM55.28a/BG505 IP-DS-MPER-TM IP chimera-DS-MPER-TM 1981 T246 CH117.4-chim_ CH117.4/BG505 IP-DS-MPER-TM IPchimera -DS-MPER-TM 1982 T247 CNE58-chim_ CNE58/BG505 P-DS-MPER-TM IPchimera -DS-MPER-TM 1983 T248 ZM53.12-chim_ ZM53.12/BG505 P-DS-MPER-TMIP chimera -DS-MPER-TM 1984 T249 ZM197.7-chim_ ZM197.7/BG505P-DS-MPER-TM IP chimera -DS-MPER-TM 1985 T250 25925-2.22-chim_25925-2.22/BG505 P-DS-MPER-TM IP chimera -DS-MPER-TM 1986 T251ZM106.9-chim_ ZM106.9/BG505 P-DS-MPER-TM-cyto IP chimera-DS-MPER-TM-cyto 1987 T252 AC10.29-chim_ AC10.29/BG505 P-DS-MPER-TM-cytoIP chimera -DS-MPER-TM-cyto 1988 T253 CH038.12-chim_ CH038.12/BG505P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1989 T254 FRO.11-chim_FRO.11/BG505 P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1990 T255QH209.14M.A2-chim_ QH209.14M.A2/BG505 P-DS-MPER-TM-cyto IP chimera-DS-MPER-TM-cyto 1991 T256 6545.V4.C1-chim_ 5545.V4.C1/BG505P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1992 T257 KER2018.11-chim_KER2018.11/BG505 P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1993 T258MB201.A1-chim_ MB201.A1/BG505 P-DS-MPER-TM-cyto IP chimera-DS-MPER-TM-cyto 1994 T259 BI369.9A-chim_ BI369.9A/BG505P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1995 T260 CNE55-chim_CNE55/BG505 P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1996 T261rH966.8-chim_ TH966.8/BG505 P-DS-MPER-TM-cyto IP chimera-DS-MPER-TM-cyto 1997 T262 R2184.c4-chim_ 32184.c4/BG505P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1998 T263 DU422.01-chim_U422.01/BG505 P-DS-MPER-TM-cyto IP chimera -DS-MPER-TM-cyto 1999 T26416055-2.3-chim_ 16055-2.3/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 2000 T265 ZM55.28a-chim_ ZM55.28a/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 2001 T266 CH117.4-chim_CH117.4/BG505 SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 2002 T267CNE58-chim_ CNE58/BG505 SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto2003 T268 ZM53.12-chim_ ZM53.12/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 2004 T269 ZM197.7-chim_ ZM197.7/BG505SOS-DS-MPER-TM-cyto SOS chimera -DS-MPER-TM-cyto 2005 T27025925-2.22-chim_ 25925-2.22/BG505 SOS-DS-MPER-TM-cyto SOS chimera-DS-MPER-TM-cyto 2006 T271 FH966.8-chim_ FH966.8/BG505 sc10ln-IP-MPER-single-chain amino sc10ln-IP chimera TM-full- acid replace-MPER-TM-full-cytoplasmic cytoplasmic domain cleavage site domainand I559P mutation 2007 T272 6545.V4.C1-chim_ 6545.V4.C1/BG505sc10ln-IP-MPER- single-chain amino sc10ln-IP chimera TM-full-acid replace -MPER-TM-full- cytoplasmic domain cleavage sitecytoplasmic domain and I559P mutation 2008 T273 R2184.c4-chim_R2184.C4/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2009 T274 ZM197.7-chim_ZM197.7/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2010 T275 ZM106.9-chim_ZM106.9/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2011 T276 ZM53.12-chim_ZM53.12/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2012 T277 CNE55-chim_CNE55/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2013 T278 DU422.01-chim_DU422.01/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full- cytoplasmic cleavage sitecytoplasmic domain domain and I559P mutation 2014 T279 25925-2.22-chim_25925-2.22/BG505 $c10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full- cytoplasmic cleavage sitecytoplasmic domain domain and I559P mutation 2015 T280 CNE58-chim_CNE58/BG505 sc10ln-IP-MPER- single-chain amino sc1Oln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2016 T281 16055-2.3-chim_16055-2.3/BG505 SC10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full- cytoplasmic cleavage sitecytoplasmic domain domain and I559P mutation 2017 T282 ZM55.28a-chim_ZM55.28a/BG505 sc10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full- cytoplasmic cleavage sitecytoplasmic domain domain and I559P mutation 2018 T283 BI369.9A-chim_BI369.9A/BG505 SC10ln-IP-MPER- single-chain amino sc10ln-IP chimeraTM-full- acid replace -MPER-TM-full-cytoplasmic cytoplasmiccleavage site domain domain and I559P mutation 2019 T284 TH966.8-chim_rH966.8/BG505 sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera-10ln-HATM 2020 T285 6545.V4.C1-chim_ 5545.V4.C1/BG505sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera -10ln-HATM 2021 T286R2184.c4-chim_ 32184.c4/BG505 sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IPchimera -10ln-HATM 2022 T287 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera -10ln-HATM 2023 T288ZM106.9-chim_ ZM106.9/BG505 sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IPchimera -10ln-HATM 2024 T289 ZM53.12-chim_ ZM53.12/BG505sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera -10ln-HATM 2025 T290R2184.c4-chim_ 32184.C4/BG505 sc10ln-IP-MPER-TM a433p sc10ln-a433p-IPchimera -MPER-TM 2026 T291 CNE55-chim_ CNE55/BG505 sc10ln-IP-10ln-HATMa433p sc10ln-a433p-IP chimera -10ln-HATM 2027 T292 6545.V4.C1-chim_5545.V4.C1/BG505 SC10ln-IP-MPER-TM a433p sc10ln-a433p-IP chimera-MPER-TM 2028 T293 DU422.01-chim_ U422.01/BG505 sc10ln-IP-10ln-HATMa433p sc10ln-a433p-IP chimera -10ln-HATM 2029 T294 25925-2.22-chim_25925-2.22/BG505 sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera-10ln-HATM 2030 T295 CNE58-chim_ CNE58/BG505 sc10ln-IP-10ln-HATM a433psc10ln-a433p-IP chimera -10ln-HATM 2031 T296 16055-2.3-chim_16055-2.3/BG505 sc10ln-IP-10ln-HATM a433p sc10ln-a433p-IP chimera-10ln-HATM 2032 T297 rH966.8-chim_ TH966.8/BG505 SC10ln-IP-MPER-TM a433psc10ln-a433p-IP chimera -MPER-TM 2033 T298 ZM55.28a-chim_ ZM55.28a/BG505sc10ln-IP-MPER-TM a433p sc10ln-a433p-IP chimera -MPER-TM 2034 T299ZM53.12-chim_ ZM53.12/BG505 sc10ln-IP-MPER-TM a433p sc10ln-a433p-IPchimera -MPER-TM 2035 T300 BI369.9A-chim_ BI369.9A/BG505sc10ln-IP-10ln-HATM a433p sc10ln-a433p--IP chimera -10ln-HATM 2036 T301ZM197.7-chim_ ZM197.7/BG505 SC10ln-IP-MPER-TM a433p sc10ln-a433p-IPchimera -MPER-TM 2037 T302 16055-2.3-chim_ 16055-2.3/BG505sc10ln-IP-MPER-TM a433p sc10ln-a433p-IP chimera -MPER-TM 2038 T303ZM55.28a-chim_ ZM55.28a/BG505 sc15ln-SOS-10ln-HATM a433psc15ln-SOS-a433p-10ln-HATM chimera 2039 T304 rH966.8-chim_ rH966.8/BG505sc10ln-IP-10ln-HATM 173-177 mutated increase the sc10ln-IP chimerato YSLFE negative charge -10ln-HATM-173-yslfe-177 in the V2 region tostabilize V2 to V3 interactions 2040 T305 6545.V4.C1-chim_6545.V4.C1/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-yslfe- 177 2041 T306R2184.c4-chim_ 32184.c4/BG505 sc10ln-IP-10ln-HATM173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-yslfe-177 2042 T307 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe-177 2043 T308 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe-177 2044 T309 ZM53.12-chim_ ZM53.12/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe-177 2045 T310 R2184.c4-chim_ 32184.C4/BG505SC10ln-IP-MPER-TM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-yslfe-177 2046 T311 CNE55-chim_ CNE55/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe-177 2047 T312 6545.V4.C1-chim_5545.V4.C1/BG505 sc10ln-IP-MPER-TM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-yslfe-177 2048 T313DU422.01-chim_ U422.01/BG505 sc10ln-IP-10ln-HATM173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-yslfe-177 2049 T314 25925-2.22-chim_ 25925-2.22/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe- 177 2050 T315 CNE58-chim_ CNE58/BG505sc10ln-IP-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-yslfe-177 2051 T316 16055-2.3-chim_16055-2.3/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-yslfe-177 2052 T317FH966.8-chim_ FH966.8/BG505 sc10ln-IP-MPER-TM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-yslfe-177 2053 T318ZM55.28a-chim_ ZM55.28a/BG505 sc10ln-IP-MPER-TM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-yslfe-177 2054 T319ZM53.12-chim_ ZM53.12/BG505 sc10ln-IP-MPER-TM 173-177 mutated to YSLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-yslfe-177 2055 T320BI369.9A-chim_ BI369.9A/BG505 sc10ln-IP-10ln-HATM173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-yslfe-177 2056 T321 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-MPER-TM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-yslfe-177 2057 T322 16055-2.3-chim_ 16055-2.3/BG505sc10ln-IP-MPER-TM 173-177 mutated to YSLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-yslfe-177 2058 T323 ZM55.28a-chim_ ZM55.28a/BG505sc15ln-SOS-DS-10ln-HATM 173-177 mutated to YSLFE Same as Seq_2039sc15ln-SOS chimera -DS-10ln-HATM-173- yslfe-177 2059 T324 FH966.8-chim_FH966.8/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-eslfe-177 2060 T3256545.V4.C1-chim_ 6545.V4.C1/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfe- 177 2061 T326 R2184.c4-chim_ 32184.C4/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2062 T327 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2063 T328 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2064 T329 ZM53.12-chim_ ZM53.12/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2065 T330 R2184.c4-chim_ 32184.C4/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfe-177 2066 T331 CNE55-chim_ CNE55/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2067 T332 6545.V4.C1-chim_5545.V4.C1/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfe-177 2068 T333DU422.01-chim_ U422.01/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfe-177 2069 T334 25925-2.22-chim_ 25925-2.22/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe- 177 2070 T335 CNE58-chim_ CNE58/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfe-177 2071 T336 16055-2.3-chim_16055-2.3/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-eslfe-177 2072 T337FH966.8-chim_ FH966.8/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfe-177 2073 T338ZM55.28a-chim_ ZM55.28a/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfe-177 2074 T339ZM53.12-chim_ ZM53.12/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFESame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfe-177 2075 T340BI369.9A-chim_ BI369.9A/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfe-177 2076 T341 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfe-177 2077 T342 16055-2.3-chim_ 16055-2.3/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFE Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfe-177 2078 T343 ZM55.28a-chim_ ZM55.28a/BG505sc15ln-SOS-DS- 173-177 mutated to ESLFE Same as Seq_2039 sc15ln-SOSchimera 10ln-HATM -DS-10ln-HATM-173- eslfe-177 2079 T344 FH966.8-chim_TH966.8/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-eslfy-177 2080 T3456545.V4.C1-chim_ 6545.V4.C1/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfy- 177 2081 T346 R2184.c4-chim_ 32184.C4/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2082 T347 ZM197.7-chim_ ZM197.7/BG505$c10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2083 T348 ZM106.9-chim_ ZM106.9/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2084 T349 ZM53.12-chim_ ZM53.12/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2085 T350 R2184.c4-chim_ 32184.c4/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfy-177 2086 T351 CNE55-chim_ CNE55/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2087 T352 6545.V4.C1-chim_5545.V4.C1/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfy-177 2088 T353DU422.01-chim_ U422.01/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfy-177 2089 T354 25925-2.22-chim_ 25925-2.22/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy- 177 2090 T355 CNE58-chim_ CNE58/BG505sc10ln-IP-10ln-HATM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -10ln-HATM-173-eslfy-177 2091 T356 16055-2.3-chim_16055-2.3/BG505 sc10ln-IP-10ln-HATM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -10ln-HATM-173-eslfy-177 2092 T357FH966.8-chim_ FH966.8/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfy-177 2093 T358ZM55.28a-chim_ ZM55.28a/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfy-177 2094 T359ZM53.12-chim_ ZM53.12/BG505 sc10ln-IP-MPER-TM 173-177 mutated to ESLFYSame as Seq_2039 sc10ln-IP chimera -MPER-TM-173-eslfy-177 2095 T360BI369.9A-chim_ BI369.9A/BG505 sc10ln-IP-10ln-HATM173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IP chimera-10ln-HATM-173-eslfy-177 2096 T361 ZM197.7-chim_ ZM197.7/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfy-177 2097 T362 16055-2.3-chim_ 16055-2.3/BG505sc10ln-IP-MPER-TM 173-177 mutated to ESLFY Same as Seq_2039 sc10ln-IPchimera -MPER-TM-173-eslfy-177 2098 T363 ZM55.28a-chim_ ZM55.28a/BG505sc15ln-SOS-DS- 173-177 mutated to ESLFY Same as Seq_2039 sc15ln-SOSchimera 10ln-HATM -DS-10ln-HATM-173- eslfy-177 2099 z 35O22 VH 2100 z35O22 VL 2101 z coat protein subunit 2102 z coat protein subunit 2103 zcoat protein subunit 2104 z coat protein subunit 2105 zcoat protein subunit 2106 z coat protein subunit 2107 zcoat protein subunit 2108 z coat protein subunit 2109 zcoat protein subunit 2110 z coat protein subunit 2111 zcoat protein subunit 2112 z coat protein subunit 2113 zcoat protein subunit 2114 0 F250-4 F250-4 2115 0 JRFL JRFL 2116 H474F2504.SOSIP.6R.201C433C F2504.SOSIP.6R. 201C433C 2117 bg505.sosip cl5ln.3g505.sosip_ 201C-433C cl5ln.201C- 433C 2118 soluble CD4(sCD4soluble CD4(sCD4 2119 BG505.SOSIP.R6.664. BG505 DNA T332NJ201C/A433C(VRC4571) 2120 BG505 WT DNA 2121 H475 *BG505.SOSIP.R6.664. BG505V1V2SOSIP; V1V2 Swap CAP256-SU SOSIP, T332N.D368R.V1V2.CAP256_ chimera664; V1V2 swap, SU R6; 201-422 disulfide 201C, stabilization,D368R, 433C, cd4 binding site KO, T332N 2122 H476 *BG505.SOSIP.R6.664.BG505 V1V2SOSIP; V1V2 Swap BB201.B42 SOSIP, T332N.D368R.V1V2.BB201.B42chimera 664; V1V2 swap, R6; 201-422 disulfide 201C, stabilization,D368R, 433C, cd4 binding site KO, T332N 2123 H477 *BG505.SOSIP.R6.664.BG505 V1V2SOSIP; V1V2 Swap KER2018.11 SOSIP, T332N.D368R.V1V2. chimera664; V1V2 swap, KER2018. R6; 201-422 disulfide 11 201C, stabilization,D368R, cd4 binding site KO, 433C, T332N 2124 H478 *BG505.SOSIP. BG505V1V2SOSIP; V1V2 Swap CH070.1 SOSIP, R6.664. chimera 664; V1V2 swap,T332N.D368R. R6; 201-422 disulfide V1V2.CH070.1 201C, stabilization,D368R, 433C, cd4 binding site KO, T332N 2125 H479 *BG505.SOSIP. BG505V1V2SOSIP; V1V2 Swap ZM233.6 SOSIP, R6.664. chimera 664; V1V2 swap, R6;201-422 disulfide 201C, stabilization, T332N.D368R. D368R, 433C,cd4 binding site KO, V1V2.ZM233.6 T332N 2126 H480 *BG505.SOSIP. BG505V1V2SOSIP; V1V2 Swap 023 SOSIP, R6.664. chimera 664; V1V2 swap,T332N.D368R.V1V2.Q23 R6; 201-422 disulfide 201C, stabilization,D368R, 433C, cd4 binding site KO, T332N 2127 H481 *BG505.SOSIP.R6.664.BG505 V1V2SOSIP; V1V2 Swap A244 SOSIP, T332N.D368R.V1V2.A244 chimera664; V1V2 swap, R6; 201-422 disulfide 201C, stabilization,D368R, 433C, T332N cd4 binding site KO, 2128 H482 *BG505.SOSIP.R6.664.BG505 V1V2SOSIP; V1V2 Swap WHO SOSIP, T332N.D368R.V1V2.WITO chimera 664;V1V2 swap, R6; 201-422 disulfide 201C, stabilization, D368R, 433C, T332Ncd4 binding site KO, 2129 H483 *BG505.SOSIP.R6.664. BG505 V1V2SOSIP;V1V2 Swap T250.4 SOSIP, T332N.D368R.V1V2.T250.4 chimera 664; V1V2 swap,R6; 201-422 disulfide 201C, stabilization, D368R, 433C, T332Ncd4 binding site KO, 2130 H484 *BG505.SOSIP.R6.664. BG505 V1V2SOSIP;V1V2 Swap CAP256-SU. SOSIP, T332N.D368R.V1V2.CAP256_ chimera 664;Week34.c1one77 V1V2 swap, SU.W34.77 R6; 201-422 disulfide 201C,stabilization, D368R, 433C, T332N cd4 binding site KO, 2131 H485*BG505.SOSIP.R6.664. BG505 V1V2SOSIP; V1V2 Swap CAP256-SU. SOSIP,T332N.D368R.V1V2.CAP256_ chimera 664; Week34.c1one80 V1V2 swap,SU.W34.80 R6; 201-422 disulfide 201C, stabilization, D368R, 433C, T332Ncd4 binding site KO, 2132 H486 *BG505.SOSIP.R6.664. BG505 V1V2SOSIP;V1V2 Swap CAP256-SU. SOSIP, T332N.D368R.V1V2.CAP256_ chimera 664;Week34.c1one81 V1V2 swap, SU.W34.781 R6; 201-422 disulfide 201C,stabilization, D368R, 433C, T332N on chimeric backbone 2133 H487*CNE58.SOSIP.R6. CNE58/BG505 VIV2SOSIP; IV2 Swap CAP256-SU SOSIP,V1V2.CAP256_SU chimera 664; V1V2 swap, R6; 201-422 disulfide 201C,stabilization, 433C on chimeric backbone 2134 H488 *CNE58.SOSIP.R6.CNE58/BG505 VIV2SOSIP; IV2 Swap BB201.B42 SOSIP, V1V2.BB201.B42 chimera664; V1V2 swap, R6; 201-422 disulfide 201C, stabilization, 433Con chimeric backbone 2135 H489 *CNE58.SOSIP.R6. CNE58/BG505 V1V2SOSIP;VIV2 Swap KER2018.11 SOSIP,  V1V2.KER2018.11 chimera 664; V1V2 swap, R6;201-422 disulfide 201C, stabilization, 433C on chimeric backbone 2136H490 *CNE58.SOSIP.R6. CNE58/BG505 V1V2SOSIP; VIV2 Swap CH070.1 SOSIP,V1V2.CH070.1 chimera 664; V1V2 swap, R6; 201-422 disulfide 201C,stabilization, 433C on chimeric backbone 2137 H491 *CNE58.SOSIP.R6.CNE58/BG505 V1V2SOSIP; VIV2 Swap ZM233.6 SOSIP, V1V2.ZM233.6 chimera664; V1V2 swap, R6; 201-422 disulfide 201C, stabilization, 433Con chimeric backbone 2138 H492 *CNE58.SOSIP.R6. CNE58/BG505 V1V2SOSIP;VIV2 Swap 023 SOSIP, V1V2.Q23 chimera 664; V1V2 swap, R6;201-422 disulfide 201C, 433C stabilization, on chimeric backbone 2139H493 *CNE58.SOSIP.R6. CNE58/BG505 V1V2SOSIP; VIV2 Swap A244 SOSIP,V1V2.A244 chimera 664; V1V2 swap, R6; 201-422 disulfide 201C,stabilization, 433C on chimeric backbone 2140 H494 *CNE58.SOSIP.R6.CNE58/BG505 V1V2SOSIP; V1V2 Swap WHO SOSIP, V1V2.WITO chimera 664;V1V2 swap, R6; 201-422 disulfide 201C, stabilization, 433Con chimeric backbone 2141 H495 *CNE58.SOSIP.R6. CNE58/BG505 V1V2SOSIP;V1V2 Swap T250.4 SOSIP, V1V2.T250.4 chimera 664; V1V2 swap, R6;201-422 disulfide 201C, stabilization, 433C on chimeric backbone 2142 D426c GenBank: KC769518.1, incorporated byreference herein as present in GenBank on Sep. 3, 2015MDAMKRGLCCVLLLCGAVFVSPSASVGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLSDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGNTTNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTDAKSSVVESNKSAVGIGAVFLGFLGAAGSTMGAASITLTVOARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKEEIWENMTWMQWDREINNYTNTIYRLLEESQNQQENNEKDLLALDSWNNLWNWFNITNWLWYIK 2143 0 426c DNA 2144 D 426c.N276D.N460D.N463DGenBank: KC769519.1, incorporated by reference herein as present inGenBank on Sep. 3, 2015MDAMKRGLCCVLLLCGAVFVSPSASVGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLNCTNVNVTSNSTNVNSSSTDNTTLGEIKNCSFDITTEIRDKTRKEYALFYRLDIVPLDNSSNPNSSNTYRLINCNTSTLTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRKDLSDNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKAIYAPPIKGNITCKSDITGLLLLRDGGDTTDNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTDAISSVVESNKSAVGIGAVFLGFLGAAGSTMGAASITLTVOARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKEEIWENMTWMQWDREINNYTNTIYRLLEESQNQQENNEKDLLALDSWNNLWNWFNITNWLWYIK 2145 D 45_01dG5 GenBank: JQ609687.1, incorporated byreference herein as present in GenBank on Sep. 3, 2015MRVMGIRKNCQRLWRGGTLFLGILMIFSAAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDYLGNATNTTSSSGGAMEGGEIKNCSFNITTSMRDKMQKEYALFYKLDVVSIDNDNASTNYRLISCNTSVITQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKAMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGLAPTRAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGWGCSGKLICTTAVPWNASWSNKSLDKIWNNMTWMEWEREINNYTGLIYNLIEESQNQQEKNEQELLELDKWASLWNWFDITKWLWYIKIFIMIVGGLVGLRIIFTVLSIVNRVRQGYSPLSFQTHLPAPRGPDRPEGIGEEGGEQRDRSDRLVTGFLAIFWVDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEILKYWWNLLQYWNQELKNSAVSLLNATAIVVAEGTDRVIEVLQRAFRAVLNIPTRIRQGLERALL 2146 H496 *426c-v1v2-WITO- BG505 platform; degly4-DS-gly504-heterologous V1V2; gly661 remainder 426c with N276, N460 andN463 glycan mutations; 201C/433C; add 504/661 sequons forglycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLOARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2147 H497 *426c-v1v2-WITO-BG505 platform; degly3-DS- heterologous V1V2; gly504-gly661remainder 426c with N276, N460 and N463 glycan mutations; 201C/433C; add504/661 sequons for glycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTSTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2148 H498 *426c-v1v2-ZM233-BG505 platform; degly4-DS-gly504- heterologous V1V2; gly661remainder 426c with N276, N460 and N463 glycan mutations; 201C/433C;add 504/661 sequons for glycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2149 H499 *426c-v1v2-ZM233- BG505 platform;degly3-DS-gly504- heterologous V1V2; gly661 remainder 426cwith N276, N460 and N463 glycan mutations; 201C/433C; add 504/661sequons for glycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTsTCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2150 H500 *d45-v1v2-WITO- BG505 platform;01dG5chim-DS- heterologous V1V2; gly504-gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylationof membrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRUNCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQsSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2151 H501 *d45-v1v2-WITO-BG505 platform; 01dG5chim-degly3- heterologous V1V2; DS-gly504-gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylation ofmembrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKUCCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2152 H502 *d45-v1v2-WITO-BG505 platform; 01dG5chim-degly4- Heterologous DS-gly504- V1V2; gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylationof membrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTaVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEWNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2153 H503 *d45-v1v2-ZM233-BG505 platform; 01dG5chim-DS- heterologous V1V2; gly504-gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylation ofmembrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2154 H504 *d45-v1v2-ZM233-BG505 platform; 01dG5chim-degly3- heterologous V1V2; DS-gly504-gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylationof membrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTOACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2155 H505 *d45-v1v2-ZM233-BG505 platform; 01dG5chim-degly4- heterologous V1V2; DS-gly504-gly661remainder 45_01dG5, 201C/433C; add 504/661 sequons for glycosylationof membrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTaVCTOACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQaSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2156 H506 *Scl5ln 426c-BG505 platform; v1v2-WITO-degly4- heterologous V1V2; DS-gly504-gly661remainder 426c with N276, N460 and N463 glycan mutations; 201C/433C,single chain format with 15 AA linker; add504/661 sequons for glycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGGGSGGGGSGGGGSGGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLOARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2157 H507*Sc10ln 426c-v1v2- BG505 platform; WITO-degly4- heterologous V1V2;DS-gly504-gly661 remainder 426c with N276, N460 and N463 glycanmutations; 201C/433C, single chain format with 10 AA linker; add504/661 sequons for glycosylation of membrane proximal regionAENLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVDQMQEDVISIWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTGYRLINCNTATCTQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIVIRSKNLADNAKIIIVQLNKSVEIVCTRPNNNTRRSIRIGPGQTFYATDIIGDIRQAYCNISGRNWSEAVNQVKKKLKEHFPHKNISFQSSSGGDLEITTHSFNCGGEFFYCNTSGLFNDTISNATIMLPCRIKQIINMWQEVGKCIYAPPIKGNITCKSDITGLLLLRDGGNTANNAEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnvtGGGSGGGGSGGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIYGLLEESQNQQEKNEQDLnAtD 2158 H508 *Scl5ln d45-BG505 platform; v1v2-ZM233- Heterologous 01dG5chim- V1V2; DS-gly504-Remainder gly661 45_01dG5; 201C/433C, single chain format with15 AA linker; add 504/661 sequons for glycosylation of membraneproximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTsVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGGGSGGGGSGGGGSGGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD 2159 H509*Sc10ln d45-v1v2- BG505 platform; ZM233-01dG5chim- HeterologousDS-gly504- V1V2; gly661 remainder 45_01dG5; 201C/433C, singlechain format with AA linker; add 504/661 sequons for glycosylation ofMembrane proximal regionAENLWVTVYYGVPVWKEATATLFCASDAKAYETEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLDCSTYNNTHNISKEMKICSFNMTTELRDKKRKVNVLFYKLDLVPLTNSSNTTNYRLISCNTSVCTQACPKISFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENIKDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGAIIGDIRQAHCNISKAKWENTLKQIARKLREHFKNETIAFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWTWNDTEVVNNTEKNINITLPCRIKQIINMWQEVGKCMYAPPIKGQIRCSSNITGLLLTRDGGSSTNGTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRnVtGGGSGGGGSGGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLnAtD

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow. Unless indicated otherwise, HIV-1 Env amino acid positions listedin the following claims correspond to the HXB2 numbering system usingthe HXB2 reference sequence set forth as SEQ ID NO: 1

1. An mRNA molecule encoding a recombinant Human Immunodeficiency Virustype 1 (HIV-1) Envelope (Env) protein comprising cysteine substitutionsat HIV-1 Env positions 201 and 433, wherein the HIV-1 Env positionscorrespond to a HXB2 reference sequence set forth as SEQ ID NO:
 1. 2.The mRNA of claim 1, wherein the cysteine substitutions to introduce anon-natural disulfide bond between at HIV-1 Env positions 201 and 433are I201C and A433C substitutions.
 3. The mRNA of claim 1, furthercomprising a methionine substitution at HIV-1 Env position 302, and aleucine substitution at HIV-1 Env position
 320. 4. The mRNA of claim 3,the methionine substitution at HIV-1 Env position 302 is a N302Msubstitution, and the leucine substitution at HIV-1 Env position 320 isaT320L substitution.
 5. The mRNA of claim 1, further comprising cysteinesubstitutions at HIV-1 Env positions 501 and 605, and a prolinesubstitution at HIV-1 Env position
 559. 6. The mRNA of claim 5, thecysteine substitutions at HIV-1 Env positions 501 and 605 are A501C andT605C substitutions; and the proline substitution at HIV-1 Env position559 is a I559P substitution.
 7. The mRNA of claim 1, further comprisingwherein a native furin cleavage site separating gp120 and gp41 subunitsof the HIV-1 Env protein is substituted with six arginine residues.
 8. Avector comprising the mRNA molecule of claim
 1. 9. An isolated host cellcomprising the vector of claim
 1. 10. An immunogenic compositioncomprising the mRNA of claim 1, and a pharmaceutically acceptablecarrier.
 11. A method for generating an immune response to HIV-1 Env ina subject, comprising administering to the subject an amount of the mRNAof claim 1 effective to generate the immune response.
 12. The method ofclaim 11, comprising a prime-boost administration of the mRNA.
 13. Themethod of claim 11, wherein the subject is at risk of or has an HIV-1infection.